Soluble fibroblast growth factor receptor 3 (sfgfr3) polypeptides and uses thereof

ABSTRACT

The invention features soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides. The invention also features methods of using sFGFR3 polypeptides to treat skeletal growth regardation disorders, such as achondroplasia.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/417,174filed on May 20, 2019, which is a continuation of U.S. application Ser.No. 15/943,436 filed on Apr. 2, 2018, now U.S. Pat. No. 10,294,289,which is a continuation of PCT International Application No.PCT/EP2017/067119, with an international filing date of Jul. 7, 2017,which claims the benefit under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 62/467,478 filed on Mar. 6, 2017, and claims the benefitunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/359,607filed on Jul. 7, 2016.

REFERENCE TO THE SEQUENCE LISTING

The application contains a Sequence Listing submitted electronically onMay 20, 2019, which is incorporated herein by reference. The SequenceListing was provided as a Text File entitled 51088-006004 SequenceListing.txt, created on May 20, 2019, and which is 105,291 bytes insize.

FIELD OF THE INVENTION

The invention features soluble fibroblast growth factor receptor 3(sFGFR3) polypeptides and compositions thereof. The invention alsofeatures methods to treat skeletal growth retardation disorders, such asachondroplasia.

BACKGROUND OF THE INVENTION

Fibroblast growth factor receptor 3 (FGFR3) is a member of thefibroblast growth factor (FGFR) family, in which there is high aminoacid sequence conservation between family members. Members of the FGFRfamily are differentiated by both ligand binding affinities and tissuedistribution. A full-length FGFR polypeptide contains an extracellulardomain (ECD), a hydrophobic transmembrane domain, and a cytoplasmictyrosine kinase domain. The ECD of FGFR polypeptides interacts withfibroblast growth factors (FGFs) to mediate downstream signaling, whichultimately influences cellular differentiation. In particular,activation of the FGFR3 protein plays a role in bone development byinhibiting chondrocyte proliferation at the growth plate and limitingbone elongation.

Gain-of-function point mutations in FGFR3 are known to cause severaltypes of human skeletal growth retardation disorders, such asachondroplasia, thanatophoric dysplasia type I (TDI), thanatophoricdysplasia type II (TDII), severe achondroplasia with developmental delayand acanthosis nigricans (SADDAN), hypochondroplasia, andcraniosynostosis syndromes (e.g., Muenke syndrome, Crouzon syndrome, andCrouzonodermoskeletal syndrome). Loss-of-function point mutations inFGFR3 are also known to cause skeletal growth retardation disorders,such as camptodactyly, tall stature, and hearing loss syndrome (CATSHL).Achondroplasia is the most common form of short-limb dwarfism and ischaracterized by disproportionate shortness of limbs and relativemacrocephaly. Approximately 97% of achondroplasia is caused by a singlepoint mutation in the gene encoding FGFR3, in which a glycine residue issubstituted with an arginine residue at position 380 of the FGFR3 aminoacid sequence. Upon ligand binding, the mutation decreases theelimination of the receptor/ligand complex resulting in prolongedintracellular signaling. This prolonged FGFR3 signaling inhibits theproliferation and differentiation of the cartilage growth plate,consequently impairing endochondral bone growth.

There exists a need for improved therapeutics that target dysfunctionalFGFR3 for treating skeletal growth retardation disorders, suchachondroplasia.

SUMMARY OF THE INVENTION

The invention features soluble fibroblast growth factor receptor 3(sFGFR3) polypeptides and uses thereof, including the use of the sFGFR3polypeptides for the treatment of skeletal growth retardation disorders(e.g., achondroplasia) in a patient (e.g., a human, particularly aninfant, a child, or an adolescent). In particular, the sFGFR3polypeptides of the invention feature a deletion of, e.g., amino acids289 to 400 of the amino acid sequence of the wildtype FGFR3 polypeptide(e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5 or32), to provide the following sFGFR3 polypeptides: sFGFR3_Del4 includingan amino acid substitution of a cysteine residue with a serine residueat position 253 (sFGFR3_Del4-C253S; SEQ ID NO: 2) and sFGFR3_Del4including an extended Ig-like C2-type domain 3 (sFGFR3_Del4-D3; SEQ IDNO: 33) and variants thereof, such as a sFGFR3 polypeptide having theamino acid sequence of SEQ ID NO: 4. Additionally, the sFGFR3polypeptides of the invention may include a signal peptide, such as asFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 18 or34.

A first aspect of the invention features a soluble fibroblast growthfactor receptor 3 (sFGFR3) polypeptide including a polypeptide sequencehaving at least 90% amino acid sequence identity (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%) sequenceidentity) to amino acid residues 23 to 357 of SEQ ID NO: 32. Inparticular, the polypeptide lacks a signal peptide (e.g., amino acids1-22 of SEQ ID NO: 32) and a transmembrane domain of FGFR3 (e.g., aminoacids of 367-399 of SEQ ID NO: 32) and (i) is less than 500 amino acidsin length (e.g., less than 475, 450, 425, 400, 375, or 350 amino acidsin length); (ii) includes 200 consecutive amino acids or fewer (e.g.,175, 150, 125, 100, 75, 50, 40, 30, 20, 15, or fewer consecutive aminoacids) of an intracellular domain of FGFR3; and/or (iii) lacks atyrosine kinase domain of FGFR3. The sFGFR3 polypeptide can also includean intracellular domain of FGFR3, such as amino acid residues 423 to 435of SEQ ID NO: 32 or an amino acid sequence having at least 90%, 92%,95%, 97%, or 99% sequence identity to amino acid residues 423 to 435 ofSEQ ID NO: 32. In particular, the polypeptide includes an amino acidsequence having at least 92%, 95%, 97%, 99%, or 100% sequence identityto SEQ ID NO: 33 (e.g., the polypeptide includes or consists of SEQ IDNO: 33). The sFGFR3 polypeptides can also include a signal peptide(e.g., the signal peptide can have the amino acid sequence of SEQ ID NO:6 or 35 or an amino acid sequence having at least 92%, 95%, 97%, or 99%sequence identity to SEQ ID NO: 6 or 35). For example, the sFGFR3polypeptide may have an amino acid sequence with at least 92%, 95%, 97%,99%, or 100% sequence identity to SEQ ID NO: 34 (e.g., the sFGFR3polypeptide includes or consists of SEQ ID NO: 34). The sFGFR3polypeptide may also have a heterologous signal peptide (e.g., thepolypeptide includes a heterologous signal peptide having the amino acidsequenceo of SEQ ID NO: 35).

A second aspect of the invention features an sFGFR3 polypeptideincluding an amino acid sequence having at least 85% sequence identity(e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity) to the amino acid sequence ofSEQ ID NO: 1, in which the sFGFR3 polypeptide further includes an aminoacid substitution that removes a cysteine residue at position 253 of SEQID NO: 1. For example, the cysteine residue at position 253 issubstituted with a serine residue or, e.g., another conservative aminoacid substitution, such as alanine, glycine, proline, or threonine. Inparticular, the sFGFR3 polypeptide includes or consists of the aminoacid sequence of SEQ ID NO: 2. For instance, the sFGFR3 polypeptide canbe an isolated sFGFR3 polypeptide. The sFGFR3 polypeptides can alsoinclude a signal peptide (e.g., the signal peptide can have the aminoacid sequence of SEQ ID NO: 6 or 35 or an amino acid sequence having atleast 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 6 or 35).For example, the sFGFR3 may have an amino acid sequence with at least92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 18 (e.g.,the sFGFR3 polypeptide includes or consists of SEQ ID NO: 18). ThesFGFR3 polypeptide may also have a heterologous signal peptide (e.g.,the polypeptide includes a heterologous signal peptide having the aminoacid sequenceo of SEQ ID NO: 35).

A third aspect of the invention features a sFGFR3 polypeptide includingan amino acid sequence having at least 85% sequence identity (e.g., atleast 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity) to the amino acid sequence of SEQ ID NO:1, in which the sFGFR3 polypeptide further includes a domain includingan amino acid sequence having at least 85% sequence identity (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity) to all or a fragment of the aminoacid sequence of SEQ ID NO: 3 (e.g., at least 10, 20, 30, 40, 45, ormore consecutive amino acids of SEQ ID NO: 3), in which the domain isinserted between amino acid residues 288 and 289 of SEQ ID NO: 1. Forexample, the domain can include an amino acid sequence having at least85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the amino acidsequence of SEQ ID NO: 3 (e.g., the domain can include or consists ofthe amino acid sequence of SEQ ID NO: 3). Optionally, the sFGFR3polypeptide includes an amino acid substitution of a cysteine residuewith a serine residue or, e.g., another conservative amino acidsubstitution, such as alanine, glycine, proline, or threonine, atposition 253 of SEQ ID NO: 1 and/or position 28 of SEQ ID NO: 3. Inparticular, the sFGFR3 polypeptide includes or consists of the aminoacid sequence of SEQ ID NO: 4. For example, the sFGFR3 polypeptide canbe an isolated sFGFR3 polypeptide.

Also featured is a polynucleotide (e.g., an isolated polynucleotide)that encodes the sFGFR3 polypeptide of the first, second, or thirdaspect of the invention including a nucleic acid sequence having atleast 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the nucleicacid sequence of SEQ ID NO: 20, 21, 36, or 37 (e.g., the polynucleotideincludes or consists of the nucleic acid of SEQ ID NO: 20, 21, 36, or37). The invention also features a vector (e.g., an isolated vector)including the polynucleotide, such as a plasmid, an artificialchromosome, a viral vector, or a phage vector. Additionally, theinvention features a host cell (e.g., an isolated host cell) includingthe polynucleotide, such as a HEK 293 cell or CHO cell.

The invention features a composition including the sFGFR3 polypeptide ofthe first, second, or third aspects of the invention or thepolynucleotide that encodes the sFGFR3 polypeptide of the first, second,or third aspects of the invention. In addition, the vector or host cellthat includes the polynucleotide encoding the sFGFR3 polypeptide can beformulated in a composition. The composition can further include apharmaceutically acceptable excipient, carrier, or diluent. Thecomposition including the sFGFR3 polypeptide, polynucleotide, or vectorcan be formulated for administration at a dose of about 0.002 mg/kg toabout 30 mg/kg, such as about 0.001 mg/kg to about 10 mg/kg. Thecomposition including the host cell can be formulated for administrationat a dose of about 1×10³ cells/mL to about 1×10¹² cells/mL. Thecomposition can be formulated for daily, weekly, or monthlyadministration, such as seven times a week, six times a week, five timesa week, four times a week, three times a week, twice a week, weekly,every two weeks, or once a month. For example, the composition includingthe sFGFR3 polypeptide, polynucleotide, or vector is formulated foradministration at a dose of about 0.25 mg/kg to about 10 mg/kg once ortwice a week. The composition can be formulated for parenteraladministration (e.g., subcutaneous administration, intravenousadministration, intramuscular administration, intra-arterialadministration, intrathecal administration, or intraperitonealadministration), enteral administration, or topical administration.Preferably, the composition is formulated for subcutaneousadministration. The invention also features a medicament that includesone or more of the compositions described above.

The invention also features a method of delivering an sFGFR3 polypeptideto tissue (e.g., skeletal tissue) in a patient (e.g. a human) having askeletal growth retardation disorder (e.g., achondroplasia) includingadministering to the patient an effective amount of the sFGFR3polypeptide of the first, second, or third aspect of the invention, apolynucleotide encoding the sFGFR3 polypeptide, a vector containing thepolynucleotide, a host cell containing the polynucleotide or vector, ora composition containing the polypeptide, polynucleotide, vector, orhost cell.

A fourth aspect of the invention features a method of treating askeletal growth retardation disorder (e.g., a FGFR3-related skeletaldisease) in a patient (e.g., a human) that includes administering thepolypeptide of the first, second, or third aspect of the invention or apolynucleotide encoding the polypeptide, a vector containing thepolynucleotide, a host cell containing the polynucleotide or vector, ora composition containing the polypeptide, polynucleotide, vector, orhost cell. The FGFR3-related skeletal disease is selected from the groupconsisting of achondroplasia, thanatophoric dysplasia type I (TDI),thanatophoric dysplasia type II (TDII), severe achondroplasia withdevelopmental delay and acanthosis nigricans (SADDAN),hypochondroplasia, a craniosynostosis syndrome (e.g., Muenke syndrome,Crouzon syndrome, and Crouzonodermoskeletal syndrome), andcamptodactyly, tall stature, and hearing loss syndrome (CATSHL). Inparticular, the skeletal growth retardation disorder is achondroplasia.

The FGFR3-related skeletal disease can be caused by expression in thepatient of a constitutively active FGFR3, such as an amino acidsubstitution of a glycine residue with an arginine residue at position380 of SEQ ID NO: 5 or 32. In particular, the patient may be diagnosedwith the skeletal growth retardation disorder (e.g., prior totreatment). For instance, the patient exhibits one or more symptoms ofthe skeletal growth retardation disorder selected from the groupconsisting of short limbs, short trunk, bowlegs, a waddling gait, skullmalformations, cloverleaf skull, craniosynostosis, wormian bones,anomalies of the hands, anomalies of the feet, hitchhiker thumb, andchest anomalies, such that the patient exhibits an improvement in theone or more symptoms of the skeletal growth retardation disorder afteradministration of the sFGFR3 polypeptide (or a polynucleotide encodingthe polypeptide, a vector containing the polynucleotide, a host cellcontaining the polynucleotide or vector, or a composition containing thepolypeptide, polynucleotide, vector, or host cell). Additionally, thepatient may have not been previously administered the sFGFR3polypeptide. For example, the patient may be an infant, a child, anadolescent, or an adult.

For example, the polypeptide is administered to the patient at a dose ofabout 0.002 mg/kg to about 30 mg/kg (e.g., a dose of about 0.001 mg/kgto about 10 mg/kg). The polypeptide may be administered to the patientone or more times daily, weekly (e.g., twice a week, three times a week,four times a week, five times a week, six times a week, or seven times aweek), every two weeks, monthly, or yearly. For example, the polypeptideis administered to the patient at a dose of about 0.25 mg/kg to about 30mg/kg at least about once or twice a week or more (e.g., the polypeptideis administered to the patient at a dose of about 2.5 mg/kg or about 10mg/kg once or twice weekly). The polypeptide can be administered to thepatient in a composition including a pharmaceutically acceptableexcipient, carrier, or diluent. The polypeptide can be administered tothe patient parenterally (e.g., subcutaneously, intravenously,intramuscularly, intra-arterially, intrathecally, or intraperitoneally),enterally, or topically. Preferably, the composition is administered tothe patient by subcutaneous injection. Additionally, the polypeptide canbind to fibroblast growth factor 1 (FGF1), fibroblast growth factor 2(FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18(FGF18), fibroblast growth factor 19 (FGF19), or fibroblast growthfactor 21 (FGF21). In particular, the binding can be characterized by anequilibrium dissociation constant (K_(d)) of about 0.2 nM to about 20nM, such as the binding is characterized by a K_(d) of about 1 nM toabout 10 nM (e.g., about 1 nm, about 2 nm, about 3 nm, about 4 nm, about5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm).The polypeptide can exhibit greater binding affinity to FGF1, FGF2,FGF9, and FGF18 relative to the binding affinity of the polypeptide toFGF19 and FGF21.

The polypeptide can have an in vivo half-life of between about 2 hoursto about 25 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 24 hours, or 25 hours). Preferably,administration of the polypeptide provides one or more, or all, of thefollowing: an increase in survival of the patient, an improvement inlocomotion of the patient, an improvement in abdominal breathing in thepatient, an increase in body and/or bone length of the patient, animprovement in the cranial ratio of the patient, and/or restoration ofthe foramen magnum shape in the patient, e.g., relative to an untreatedpatient (e.g., an untreated achondroplasia patient).

The invention also features a method of producing the sFGFR3 polypeptideof the first, second, or third aspect of the invention, which includesculturing the host cell described above (e.g., a CHO cell or HEK 293cell) in a culture medium under conditions suitable to effect expressionof the sFGFR3 polypeptide and recovering the sFGFR3 polypeptide from theculture medium. In particular, the recovering includes chromatography,such as affinity chromatography (e.g., ion exchange chromatography oranti-FLAG chromatography, such as immunoprecipitation) or size exclusionchromatography.

A fifth aspect of the invention features the polypeptide of the first,second, or third aspect of the invention (or a polynucleotide encodingthe polypeptide, a vector containing the polynucleotide, a host cellcontaining the polynucleotide or vector, or a composition containing thepolypeptide, polynucleotide, vector, or host cell) for treating askeletal growth retardation disorder in a patient. In particular, thesFGFR3 polypeptide can bind to fibroblast growth factor 1 (FGF1),fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9),fibroblast growth factor 18 (FGF18), fibroblast growth factor 19(FGF19), or fibroblast growth factor 21 (FGF21).

A sixth aspect of the invention features a sFGFR3 polypeptide (or apolynucleotide encoding the polypeptide, a vector containing thepolynucleotide, a host cell containing the polynucleotide or vector, ora composition containing the polypeptide, polynucleotide, vector, orhost cell) including an amino acid sequence having at least 85% sequenceidentity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acidsequence of SEQ ID NO: 1 for treating a skeletal growth retardationdisorder in a patient (e.g., a human), in which the sFGFR3 polypeptidefurther includes an amino acid substitution that removes a cysteineresidue at position 253 of SEQ ID NO: 1. For example, the cysteineresidue at position 253 is substituted with a serine residue or, e.g.,another conservative amino acid substitution, such as alanine, glycine,proline, or threonine. In particular, the sFGFR3 polypeptide includes orconsists of the amino acid sequence of SEQ ID NO: 2. For example, thesFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. Furthermore,the sFGFR3 polypeptide can bind to fibroblast growth factor 1 (FGF1),fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9),fibroblast growth factor 18 (FGF18), fibroblast growth factor 19(FGF19), or fibroblast growth factor 21 (FGF21).

A seventh aspect of the invention features a sFGFR3 polypeptide (or apolynucleotide encoding the polypeptide, a vector containing thepolynucleotide, a host cell containing the polynucleotide or vector, ora composition containing the polypeptide, polynucleotide, vector, orhost cell) including an amino acid sequence having at least 85% sequenceidentity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acidsequence of SEQ ID NO: 1 for treating a skeletal growth retardationdisorder in a patient (e.g., a human), in which the sFGFR3 polypeptidefurther includes a domain including an amino acid sequence having atleast 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity) to all or a fragment of the amino acid sequence of SEQ ID NO:3 (e.g., at least 10, 20, 30, 40, 45, or more consecutive amino acids ofSEQ ID NO: 3), in which the domain is inserted between amino acidresidues 288 and 289 of SEQ ID NO: 1. For example, the domain caninclude an amino acid sequence having at least 85%, 90%, 92%, 95%, 97%,or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3(e.g., the domain can include or consists of the amino acid sequence ofSEQ ID NO: 3). Optionally, the sFGFR3 polypeptide includes an amino acidsubstitution of a cysteine residue with a serine residue or, e.g.,another conservative amino acid substitution, such as alanine, glycine,proline, or threonine, at position 253 of SEQ ID NO: 1 and/or position28 of SEQ ID NO: 3. In particular, the sFGFR3 polypeptide includes orconsists of the amino acid sequence of SEQ ID NO: 4. For example, thesFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. Furthermore,the sFGFR3 polypeptide can bind to fibroblast growth factor 1 (FGF1),fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9),fibroblast growth factor 18 (FGF18), fibroblast growth factor 19(FGF19), or fibroblast growth factor 21 (FGF21).

The use of the fifth, sixth, or seventh aspect also features theadministration of a polynucleotide, vector, host cell, or composition ofthe first, second, or third aspect of the invention. The sFGFR3polypeptide of the sixth aspect of the invention can be encoded by apolynucleotide including a nucleic acid sequence having at least 85%,90%, 92%, 95%, 97%, or 99% sequence identity to the nucleic acidsequence of SEQ ID NO: 20 or 36 (e.g., the polynucleotide includes orconsists of the nucleic acid of SEQ ID NO: 20 or 36). The sFGFR3polypeptide of the fifth or seventh aspect of the invention can beencoded by a polynucleotide including a nucleic acid sequence having atleast 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the nucleicacid sequence of SEQ ID NO: 21 or 37 (e.g., the polynucleotide includesor consists of the nucleic acid of SEQ ID NO: 21 or 37).

The skeletal growth retardation disorder of the fifth, sixth, or seventhaspect of the invention can be any FGFR3-related skeletal disease, suchas achondroplasia, TDI, TDII, severe achondroplasia with developmentaldelay and acanthosis nigricans (SADDAN), hypochondroplasia, acraniosynostosis syndrome (e.g., Muenke syndrome, Crouzon syndrome, andCrouzonodermoskeletal syndrome), or CATSHL. In particular, the skeletalgrowth retardation disorder is achondroplasia. The FGFR3-relatedskeletal disease can be caused by expression in the patient of aconstitutively active FGFR3, e.g., in which the constitutively activeFGFR3 includes an amino acid substitution of a glycine residue with anarginine residue at position 380 of SEQ ID NO: 5.

The patient (e.g., a human) of the fifth, sixth, or seventh aspect ofthe invention can be one that has been diagnosed with the skeletalgrowth retardation disorder (e.g., prior to treatment). The patient canexhibit one or more symptoms of the skeletal growth retardation disorder(e.g., achondroplasia) selected from the group consisting of shortlimbs, short trunk, bowlegs, a waddling gait, skull malformations,cloverleaf skull, craniosynostosis, wormian bones, anomalies of thehands, anomalies of the feet, hitchhiker thumb, and chest anomalies. Asa result of the methods, the patient can exhibit an improvement in theone or more symptoms of the skeletal growth retardation disorder afteradministration of the sFGFR3 polypeptide. Moreover, administration ofthe sFGFR3 polypeptide can increase survival of the patient and/orrestore the shape of the foramen magnum of the patient. The patient canbe an infant, a child, an adolescent, or an adult. Additionally, thepatient can be one that has not been previously administered the sFGFR3polypeptide (or a polynucleotide encoding the polypeptide, a vectorcontaining the polynucleotide, a host cell containing the polynucleotideor vector, or a composition containing the polypeptide, polynucleotide,vector, or host cell).

The sFGFR3 polypeptide, polynucleotide, or vector of the fifth, sixth,or seventh aspect of the invention can be administered to the patient(e.g., a human) at a dose of about 0.002 mg/kg to about 30 mg/kg, suchas about 0.001 mg/kg to about 10 mg/kg. The composition including thehost cell of the fourth or fifth aspect of the invention can beadministered to the patient (e.g., a human) at a dose of about 1×10³cells/mL to about 1×10¹² cells/mL. For example, the sFGFR3 polypeptide,polynucleotide, vector, or host cell is administered to the patient oneor more times daily, weekly, monthly, or yearly (e.g., the sFGFR3polypeptide is administered to the patient seven times a week, six timesa week, five times a week, four times a week, three times a week, twicea week, weekly, every two weeks, or once a month). In particular, thesFGFR3 polypeptide is administered to the patient at a dose of about0.25 mg/kg to about 10 mg/kg once or twice a week. The sFGFR3polypeptide can be administered to the patient in a compositionincluding a pharmaceutically acceptable excipient, carrier, or diluent.For example, the composition is administered to the patient parenterally(e.g., subcutaneously, intravenously, intramuscularly, intra-arterially,intrathecally, or intraperitoneally), enterally, or topically. Inparticular, the composition is administered to the patient bysubcutaneous injection.

The invention features a method of manufacturing the sFGFR3 polypeptideof the first aspect of the invention by deleting the signal peptide, thetransmembrane domain, and a portion of the intracellular domain from aFGFR3 polypeptide (e.g., to manufacture a polypeptide having the aminoacid sequence of SEQ ID NO: 33). In particular, the intracellular domainconsists of amino acid residues 436 to 806 of SEQ ID NO: 32. Theinvention also features a method of manufacturing the sFGFR3 polypeptideof the second aspect of the invention by introducing an amino acidsubstitution that removes a cysteine residue at position 253 of SEQ IDNO: 1 (e.g., to manufacture a polypeptide having the amino acid sequenceof SEQ ID NO: 2). For example, the cysteine residue at position 253 issubstituted with a serine residue or, e.g., another conservative aminoacid substitution, such as alanine, glycine, proline, or threonine.

The invention also features a kit including the sFGFR3 polypeptide ofthe first, second, or third aspect of the invention (e.g., a polypeptidehaving the amino acid sequence of SEQ ID NO: 2, 4, or 33), thepolynucleotide of the first, second, or third aspect of the invention(e.g., a polynucleotide having the nucleic acid sequence of SEQ ID NO:20, 21, 36, or 37), the vector of the first, second, or third aspect ofthe invention (e.g., a plasmid, an artificial chromosome, a viralvector, or a phage vector), or the host cell of the first, second, orthird aspect of the invention (e.g., a HEK 293 cell or a CHO cell), inwhich the kit optionally includes instructions for using the kit.

Definitions

As used herein, “a” and “an” means “at least one” or “one or more”unless otherwise indicated. In addition, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise.

As used herein, “about” refers to an amount that is ±10% of the recitedvalue and is preferably ±5% of the recited value, or more preferably ±2%of the recited value. For instance, the term “about” can be used tomodify all dosages or ranges recited herein by ±10% of the recitedvalues or range endpoints.

The term “domain” refers to a conserved region of the amino acidsequence of a polypeptide (e.g. a FGFR3 polypeptide) having anidentifiable structure and/or function within the polypeptide. A domaincan vary in length from, e.g., about 20 amino acids to about 600 aminoacids. Exemplary domains include the immunoglobulin domains of FGFR3(e.g., Ig-like C2-type domain 1, Ig-like C2-type domain 2, and Ig-likeC2-type domain 3).

The term “dosage” refers to a determined quantity of an active agent(e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptidehaving the amino acid sequence of SEQ ID NO: 2, 4, or 33) calculated toproduce a desired therapeutic effect (e.g., treatment of a skeletalgrowth retardation disorder, such as achondroplasia) when the activeagent is administered to a patient (e.g., a patient having a skeletalgrowth retardation disorder, such as achondroplasia). A dosage may bedefined in terms of a defined amount of the active agent or a definedamount coupled with a particular frequency of administration. A dosageform can include an sFGFR3 polypeptide or fragment thereof inassociation with any suitable pharmaceutical excipient, carrier, ordiluent.

The terms “effective amount,” “amount effective to,” and“therapeutically effective amount” refer to an amount of an sFGFR3polypeptide, a vector encoding a sFGR3, and/or an sFGFR3 compositionthat is sufficient to produce a desired result, for example, thetreatment of a skeletal growth retardation disorder (e.g.,achondroplasia).

The terms “extracellular domain” and “ECD” refer to the portion of aFGFR3 polypeptide that extends beyond the transmembrane domain into theextracellular space. The ECD mediates binding of a FGFR3 to one or morefibroblast growth factors (FGFs). For instance, an ECD includes theIg-like C2-type domains 1-3 of a FGFR3 polypeptide. In particular, theECD includes the Ig-like C2-type domain 1 of a wildtype (wt) FGFR3polypeptide (e.g., amino acids 36-88 of a wt FGFR3 polypeptide havingthe amino acid sequence of SEQ ID NO: 5 (a mature FGFR3 protein withouta signal sequence) or amino acids 57-110 of a wt FGFR3 polypeptidehaving the amino acid sequence of SEQ ID NO: 32 (a precursor FGFR3protein with the signal sequence)), the Ig-like C2-type domain 2 of awildtype (wt) FGFR3 polypeptide (e.g., amino acids 139-234 of a wt FGFR3polypeptide having the amino acid sequence of SEQ ID NO: 5 or aminoacids 161-245 of a wt FGFR3 polypeptide having the amino acid sequenceof SEQ ID NO: 32), and the Ig-like C2-type domain 3 of a wt FGFR3polypeptide (e.g., amino acids 247-335 of a wt FGFR3 polypeptide havingthe amino acid sequence of SEQ ID NO: 5 or amino acids 268-310 of a wtFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32). AnECD of a FGFR3 can also include a fragment of the wildtype FGFR3 Ig-likeC2-type domain 3, for instance, aa 247-288 of SEQ ID NO: 1, which canfurther include, e.g., an amino acid substitution of a cysteine residuewith a serine residue or another conservative amino acid substitution(e.g., alanine, glycine, proline, or threonine) at position 253 of SEQID NO: 1 (e.g., aa 247-288 of SEQ ID NO: 2). Additionally, an ECD caninclude an Ig-like C2-type domain 3 of, e.g., aa 247-335 of SEQ ID NO:4. Thus, exemplary EGDs of FGFR3 polypeptides include, e.g., thosepolypeptides having the amino acid sequence of aa 1-288 of SEQ ID NOs: 1and 2 or aa 1-335 of SEQ ID NOs: 4 and 33. In particular, the ECD of aFGFR3 polypeptide includes aa 1-335 of SEQ ID NO: 33.

The term “FGFR3-related skeletal disease,” as used herein, refers to askeletal disease that is caused by an abnormal increase in theactivation of FGFR3, such as by expression of a gain-of-function mutantof the FGFR3. The phrase “gain-of-function mutant of the FGFR3” refersto a mutant of the FGFR3 exhibiting a biological activity, such astriggering downstream signaling, which is higher than the biologicalactivity of the corresponding wild-type FGFR3 (e.g., a polypeptidehaving the amino acid sequence of SEQ ID NO: 5) in the presence of a FGFligand. FGFR3-related skeletal diseases can include an inherited or asporadic disease. Exemplary FGFR3-related skeletal diseases include, butare not limited to, achondroplasia, thanatophoric dysplasia type I(TDI), thanatophoric dysplasia type II (TDII), severe achondroplasiawith developmental delay and acanthosis nigricans (SADDAN),hypochondroplasia, a craniosynostosis syndrome (e.g., Muenke syndrome,Crouzon syndrome, and Crouzonodermoskeletal syndrome), andcamptodactyly, tall stature, and hearing loss syndrome (CATSHL).

The terms “fibroblast growth factor” and “FGF” refer to a member of theFGF family, which includes structurally related signaling moleculesinvolved in various metabolic processes, including endocrine signalingpathways, development, wound healing, and angiogenesis. FGFs play keyroles in the proliferation and differentiation of a wide range of celland tissue types. The term preferably refers to FGF1, FGF2, FGF9, FGF18,FGF19, and FGF21, which have been shown to bind FGFR3. For instance,FGFs can include human FGF1 (e.g., a polypeptide having the amino acidsequence of SEQ ID NO: 13), human FGF2 (e.g., a polypeptide having theamino acid sequence of SEQ ID NO: 14), human FGF9 (e.g., a polypeptidehaving the amino acid sequence of SEQ ID NO: 15), human FGF18 (e.g., apolypeptide having the amino acid sequence of SEQ ID NO: 16), humanFGF19 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:38), and human FGF21 (e.g., a polypeptide having the amino acid sequenceof SEQ ID NO: 39).

The terms “fibroblast growth factor receptor 3,” “FGFR3,” or “FGFR3receptor,” as used herein, refer to a polypeptide that specificallybinds one or more FGFs (e.g., FGF1, FGF2, FGF9, FGF18, FGF19, and/orFGF21). The human FGFR3 gene, which is located on the distal short armof chromosome 4, encodes an 806 amino acid protein precursor (fibroblastgrowth factor receptor 3 isoform 1 precursor), which contains 19 exons,and includes a signal peptide (e.g., a polypeptide having the amino acidsequence of SEQ ID NO: 6 or 35). Mutations in the FGFR3 amino acidsequence that lead to skeletal growth disorders, (e.g., achondroplasia),include, e.g., the substitution of a glycine residue at position 380with an arginine residue (i.e., G380R). The naturally occurring humanFGFR3 gene has a nucleotide sequence as shown in Genbank Accessionnumber NM 000142.4 and the naturally occurring human FGFR3 protein hasan amino acid sequence as shown in Genbank Accession number NP 000133,herein represented by SEQ ID NO: 5. The wildtype FGFR3 (e.g., apolypeptide having the amino acid sequence of SEQ ID NO: 5) consists ofan extracellular immunoglobulin-like membrane domain including Ig-likeC2-type domains 1-3 (amino acid residues 1 to 335), a transmembranedomain (amino acid residues 345 to 377), and an intracellular domain(amino acid residues 378 to 784). FGFR3s can include fragments and/orvariants (e.g., splice variants, such as splice variants utilizingalternate exon 8 rather than exon 9) of the full-length, wild-type FGFR3(e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5).

The terms “fragment” and “portion” refer to a part of a whole, such as apolypeptide or nucleic acid molecule that contains, preferably, at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or more of the entire length of the reference nucleic acidmolecule or polypeptide. A fragment or portion may contain, e.g., 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 500, 600, 700, or more amino acid residues, up to the entirelength of the reference polypeptide (e.g., a polypeptide having theamino acid sequence of SEQ ID NO: 5 or 32). For example, a FGFR3fragment can include any polypeptide having at least 200, 205, 210, 215,220, 225, 235, 230, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290, or300 consecutive amino acids of SEQ ID NO: 1 or 2. Additionally, a FGFR3fragment can include any polypeptide having at least 200, 205, 210, 215,220, 225, 235, 230, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290,300, 305, 310, 315, 320, 325, 330, 335, 345, or 345 consecutive aminoacids of SEQ ID NO: 4 or 33.

As used herein, the term “host cell” refers to a vehicle that includesthe necessary cellular components, e.g., organelles, needed to expressan sFGFR3 polypeptide from a corresponding polynucleotide. The nucleicacid sequence of the polynucleotide is typically included in a nucleicacid vector (e.g., a plasmid, an artificial chromosome, a viral vector,or a phage vector) that can be introduced into the host cell byconventional techniques known in the art (e.g., transformation,transfection, electroporation, calcium phosphate precipitation, anddirect microinjection). A host cell may be a prokaryotic cell, e.g., abacterial or an archaeal cell, or a eukaryotic cell, e.g., a mammaliancell (e.g., a Chinese Hamster Ovary (CHO) cell or a Human EmbryonicKidney 293 (HEK 293)). Preferably, the host cell is a mammalian cell,such as a CHO cell.

By “isolated” is meant separated, recovered, or purified from itsnatural environment. For example, an isolated sFGFR3 polypeptide (e.g.,an sFGFR3 polypeptide or variant thereof, such as a polypeptide havingthe amino acid sequence of SEQ ID NO: 2 or 4) can be characterized by acertain degree of purity after isolating the sFGFR3 polypeptide from,e.g., cell culture media. An isolated sFGFR3 polypeptide can be at least75% pure, such that the sFGFR3 polynucleotide constitutes at least 75%by weight of the total material (e.g., polypeptides, polynucleotides,cellular debris, and environmental contaminants) present in thepreparation: (e.g., at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 99%, or at least 99.5% byweight of the total material present in the preparation). Likewise, anisolated polynucleotide encoding an sFGFR3 polypeptide (e.g., apolynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21,36, or 37), or an isolated host cell (e.g., CHO cell, a HEK 293 cell, Lcell, C127 cell, 3T3 cell, BHK cell, or COS-7 cell) can be at least 75%pure, such that the polynucleotide or host cell constitutes at least 75%by weight of the total material (e.g., polypeptides, polynucleotides,cellular debris, and environmental contaminants) present in thepreparation (e.g., at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 99%, or at least 99.5% byweight of the total material present in the preparation).

“Polynucleotide” and “nucleic acid molecule,” as used interchangeablyherein, refer to polymers of nucleotides of any length, and include DNAand RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or analogs thereof, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase or by asynthetic reaction. A polynucleotide can include modified nucleotides,such as methylated nucleotides and analogs thereof. If present,modification to the nucleotide structure can be imparted before or afterassembly of the polymer. The sequence of nucleotides can be interruptedby non-nucleotide components. A polynucleotide can be further modifiedafter synthesis, such as by conjugation with a label.

The terms “patient” and “subject” refer to a mammal, including, but notlimited to, a human (e.g., a human having a skeletal growth retardationdisorder, such as achondroplasia) or a non-human mammal (e.g., anon-human mammal having a skeletal growth retardation disorder, such asachondroplasia), such as a bovine, equine, canine, ovine, or feline.Preferably, the patient is a human having a skeletal growth retardationdisorder (e.g., achondroplasia), particularly an infant, a child, or anadolescent having a skeletal growth retardation disorder (e.g.,achondroplasia).

The terms “parenteral administration,” “administered parenterally,” andother grammatically equivalent phrases, as used herein, refer to a modeof administration of compositions including an sFGFR3 polypeptide (e.g.,an sFGFR3 polypeptide or variant thereof, such as a polypeptide havingthe amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3polypeptide including a signal peptide, such as a polypeptide having theamino acid sequence of SEQ ID NO: 18 or 34) other than enteral andtopical administration, usually by injection, and include, withoutlimitation, subcutaneous, intradermal, intravenous, intranasal,intraocular, pulmonary, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary,intraperitoneal, transtracheal, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, epidural, intracerebral,intracranial, intracarotid, and intrasternal injection and infusion.

By “pharmaceutically acceptable diluent, excipient, carrier, oradjuvant” is meant a diluent, excipient, carrier, or adjuvant,respectively that is physiologically acceptable to the subject (e.g., ahuman) while retaining the therapeutic properties of the pharmaceuticalcomposition (e.g., an sFGFR3 polypeptide or variant thereof, such as apolypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, ora sFGFR3 polypeptide including a signal peptide, such as a polypeptidehaving the amino acid sequence of SEQ ID NO: 18 or 34) with which it isadministered. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable diluents,excipients, carriers, or adjuvants and their formulations are known toone skilled in the art.

By “pharmaceutical composition” is meant a composition containing anactive agent, such as an sFGFR3 (e.g., an sFGFR3 polypeptide or variantthereof, such as a polypeptide having the amino acid sequence of SEQ IDNO: 2, 4, or 33, or a sFGFR3 polypeptide including a signal peptide,such as a polypeptide having the amino acid sequence of SEQ ID NO: 18 or34), formulated with at least one pharmaceutically acceptable excipient,carrier, or diluent. The pharmaceutical composition may be manufacturedor sold with the approval of a governmental regulatory agency as part ofa therapeutic regimen for the treatment of a disease or event (e.g., askeletal growth retardation disorder, such achondroplasia) in a patient(e.g., a patient having a skeletal growth retardation disorder, such asa patient having achondroplasia). Pharmaceutical compositions can beformulated, e.g., for parenteral administration, such as forsubcutaneous administration (e.g. by subcutaneous injection) orintravenous administration (e.g., as a sterile solution free ofparticulate emboli and in a solvent system suitable for intravenoususe), or for oral administration (e.g., as a tablet, capsule, caplet,gelcap, or syrup).

As used herein, the term “sequence identity” refers to the percentage ofamino acid (or nucleic acid) residues of a candidate sequence, e.g., anFGFR3 polypeptide, that are identical to the amino acid (or nucleicacid) residues of a reference sequence, e.g., a wild-type sFGFR3polypeptide (e.g., a polypeptide having the amino acid sequence of SEQID NO: 5 or 32) or an sFGFR3 polypeptide (e.g., an sFGFR3 polypeptide orvariant thereof, such as a polypeptide having the amino acid sequence ofSEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide including a signalpeptide, such as a polypeptide having the amino acid sequence of SEQ IDNO: 18 or 34), after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent identity (e.g., gaps can beintroduced in one or both of the candidate and reference sequences foroptimal alignment and non-homologous sequences can be disregarded forcomparison purposes). Alignment for purposes of determining percentidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software, suchas BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN,ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Those skilled in theart can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. For instance, the percentamino acid (or nucleic acid) sequence identity of a given candidatesequence to, with, or against a given reference sequence (which canalternatively be phrased as a given candidate sequence that has orincludes a certain percent amino acid (or nucleic acid) sequenceidentity to, with, or against a given reference sequence) is calculatedas follows:

100×(fraction of A/B)

where A is the number of amino acid (or nucleic acid) residues scored asidentical in the alignment of the candidate sequence and the referencesequence, and where B is the total number of amino acid (or nucleicacid) residues in the reference sequence. In particular, a referencesequence aligned for comparison with a candidate sequence can show thatthe candidate sequence exhibits from, e.g., 50% to 100% identity acrossthe full length of the candidate sequence or a selected portion ofcontiguous amino acid (or nucleic acid) residues of the candidatesequence. The length of the candidate sequence aligned for comparisonpurpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%,70%, 80%, 90%, or 100% of the length of the reference sequence. When aposition in the candidate sequence is occupied by the same amino acid(or nucleic acid) residue as the corresponding position in the referencesequence, then the molecules are identical at that position.

By “signal peptide” is meant a short peptide (e.g., 5-30 amino acids inlength, such as 22 amino acids in length) at the N-terminus of apolypeptide that directs a polypeptide towards the secretory pathway(e.g., the extracellular space). The signal peptide is typically cleavedduring secretion of the polypeptide. The signal sequence may direct thepolypeptide to an intracellular compartment or organelle, e.g., theGolgi apparatus. A signal sequence may be identified by homology, orbiological activity, to a peptide with the known function of targeting apolypeptide to a particular region of the cell. One of ordinary skill inthe art can identify a signal peptide by using readily availablesoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOXprograms). A signal peptide can be one that is, for example,substantially identical to the amino acid sequence of SEQ ID NO: 6 or35.

The term “skeletal growth retardation disorder,” as used herein, refersto a skeletal disease characterized by deformities and/or malformationsof the bones. These disorders include, but are not limiting to, skeletalgrowth retardation disorders caused by growth plate (physeal) fractures,idiopathic skeletal growth retardation disorders, or FGFR3-relatedskeletal diseases. In particular, a patient having a skeletal growthretardation disorder (e.g., achondroplasia) may have bones that areshorter than the bones of a healthy patient. For example, the skeletalgrowth retardation disorder may include a skeletal dysplasia, e.g.,achondroplasia, homozygous achondroplasia, heterozygous achondroplasia,achondrogenesis, acrodysostosis, acromesomelic dysplasia,atelosteogenesis, camptomelic dysplasia, chondrodysplasia punctata,rhizomelic type of chondrodysplasia punctata, cleidocranial dysostosis,congenital short femur, craniosynostosis (e.g., Muenke syndrome, Crouzonsyndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, orCrouzonodermoskeletal syndrome), dactyly, brachydactyly, camptodactyly,polydactyly, syndactyly, diastrophic dysplasia, dwarfism, dyssegmentaldysplasia, enchondromatosis, fibrochondrogenesis, fibrous dysplasia,hereditary multiple exostoses, hypochondroplasia, hypophosphatasia,hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Kniest dysplasia,Kniest syndrome, Langer-type mesomelic dysplasia, Marfan syndrome,McCune-Albright syndrome, micromelia, metaphyseal dysplasia, Jansen-typemetaphyseal dysplasia, metatrophic dysplasia, Morquio syndrome,Nievergelt-type mesomelic dysplasia, neurofibromatosis, osteoarthritis,osteochondrodysplasia, osteogenesis imperfecta, perinatal lethal type ofosteogenesis imperfecta, osteopetrosis, osteopoikilosis, peripheraldysostosis, Reinhardt syndrome, Roberts syndrome, Robinow syndrome,short-rib polydactyly syndromes, short stature, spondyloepiphysealdysplasia congenita, and spondyloepimetaphyseal dysplasia.

The terms “soluble fibroblast growth factor receptor 3,” “solubleFGFR3,” and “sFGFR3” refer to a FGFR3 that is characterized by theabsence or functional disruption of all or a substantial part of thetransmembrane domain and any polypeptide portion that would anchor theFGFR3 polypeptide to a cell membrane (e.g., a tyrosine kinase domain).An sFGFR3 polypeptide is a non-membrane bound form of an FGFR3polypeptide. In particular, the transmembrane domain of FGFR3 extendsfrom amino acid residues 345 to 377 of the wild-type FGFR3 sequence(e.g, a polypeptide having the amino acid sequence of SEQ ID NO: 5) oramino acid residues 367 to 399 of the wild-type FGFR3 sequence includinga signal peptide (e.g., a polypeptide having the amino acid sequence ofSEQ ID NO: 32). Thus, the sFGFR3 polypeptide can include a deletion of aportion or all of amino acid residues 345 to 377 of the wild-type FGFR3polypeptide sequence (e.g., a polypeptide having the amino acid sequenceof SEQ ID NO: 5) or amino acid residues 367 to 399 of the wild-typeFGFR3 sequence including a signal peptide (e.g., a polypeptide havingthe amino acid sequence of SEQ ID NO: 32). The sFGFR3 polypeptide canfurther include deletions of the cytoplasmic domain of the wild-typeFGFR3 polypeptide sequence (amino acid residues 378 to 784 of SEQ ID NO:5) or the wild-type FGFR3 polypeptide sequence including a signalpeptide sequence (amino acid residues 378 to 806 of SEQ ID NO: 32).

Exemplary sFGFR3 polypeptides can include, but are not limited to, atleast amino acids 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 230, 1 to 235, 1 to240, 1 to 245, 1 to 250, 1 to 252, 1 to 255, 1 to 260, 1 to 265, 1 to270, 1 to 275, 1 to 280, 1 to 285, 1 to 290, 1 to 295, or 1 to 300, or 1to 301 of SEQ ID NOs: 1 or 2. sFGFR3 polypeptides can include anypolypeptide having at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more) sequence identity to any of these sFGFR3 polypeptides ofSEQ ID NO: 1 or 2. Additionally, exemplary sFGFR3 polypeptides caninclude, but are not limited to, at least amino acids 1 to 100, 1 to125, 1 to 150, 1 to 175, 1 to 200, 1 to 205, 1 to 210, 1 to 215, 1 to220, 1 to 225, 1 to 230, 1 to 235, 1 to 240, 1 to 245, 1 to 250, 1 to255, 1 to 260, 1 to 265, 1 to 270, 1 to 275, 1 to 280, 1 to 285, 1 to290, 1 to 295, 1 to 300, 1 to 305, 1 to 310, 1 to 315, 1 to 320, 1 to325, 1 to 330, 1 to 335, 1 to 340, 1 to 345, or 1 to 348 of SEQ ID NO: 4or 33. sFGFR3 polypeptides can include any polypeptide having at least50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identityto any of these sFGFR3 polypeptides having the amino acid sequence ofSEQ ID NO: 4 or 33. Any of the above sFGFR3 polypeptides or variantsthereof can optionally include a signal peptide at the N-terminalposition, such as amino acids 1 to 22 of SEQ ID NO: 6(MGAPACALALCVAVAIVAGASS) or amino acids 1 to 19 of SEQ ID NO: 35 (e.g.,MMSFVSLLLVGILFHATQA).

By “treating” and “treatment” is meant a reduction (e.g., by at leastabout 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 99%, or even 100%) in the progression or severity of askeletal growth retardation disorder (e.g., achondroplasia), or in theprogression, severity, or frequency of one or more symptoms of askeletal growth retardation disorder (e.g., achondroplasia) in a patient(e.g., a human, such as an infant, a child, or an adolescent). Treatmentcan occur for a treatment period, in which an sFGFR3 polypeptide isadministered for a period of time (e.g., days, months, years, or longer)to treat a patient (e.g., a human, such as an infant, a child, or anadolescent) having a skeletal growth retardation disorder, such asachondroplasia. Exemplary symptoms of achondroplasia that can be treatedwith an sFGFR3 (e.g., an sFGFR3 polypeptide or variant thereof, such asa polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33,or a sFGFR3 polypeptide including a signal peptide, such as apolypeptide having the amino acid sequence of SEQ ID NO: 18 or 34)include, but are not limited to, short stature, a long trunk, shortenedlimbs, an adult height of between about 42 to about 56 inches, arelatively large head, a forehead that is prominent, underdevelopedportions of the face, genu valgum (e.g., “knock-knee”), a waddling gait,short and stubby fingers, short and stubby toes, limited ability tostraighten the arm at the elbow, an excessive curve of the lower back,dental problems (e.g. from overcrowding of teeth), weight controlproblems, neurological problems, respiratory problems, and/or pain andnumbness in the lower back and/or spine.

The term “variant,” with respect to a polypeptide, refers to apolypeptide (e.g., an sFGFR3 polypeptide or variant thereof, such as apolypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, ora sFGFR3 polypeptide including a signal peptide, such as a polypeptidehaving the amino acid sequence of SEQ ID NO: 18 or 34) that differs byone or more changes in the amino acid sequence from the polypeptide fromwhich the variant is derived (e.g., the parent polypeptide, such apolypeptide having the amino acid sequence of SEQ ID NO: 1 or 7). Theterm “variant,” with respect to a polynucleotide, refers to apolynucleotide (e.g., a polynucleotide encoding a sFGFR3 polypeptide,such as a polynucleotide having the nucleic acid sequence of SEQ ID NO:20, 21, 36, or 37) that differs by one or more changes in the nucleicacid sequence from the polynucleotide from which the variant is derived(e.g., the parent polynucleotide). The changes in the amino acid ornucleic acid sequence of the variant can be, e.g., amino acid or nucleicacid substitutions, insertions, deletions, N-terminal truncations, orC-terminal truncations, or any combination thereof. In particular, theamino acid substitutions may be conservative and/or non-conservativesubstitutions. A variant can be characterized by amino acid sequenceidentity or nucleic acid sequence identity to the parent polypeptide(e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptidehaving the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3polypeptide including a signal peptide, such as a polypeptide having theamino acid sequence of SEQ ID NO: 18 or 34) or parent polynucleotide(e.g., a polynucleotide encoding a sFGFR3 polypeptide, such as apolynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21,36, or 37), respectively. For example, a variant can include anypolypeptide having at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more) sequence identity to a polypeptide having the amino acidsequence of SEQ ID NO: 1, 2, 4, or 33. A variant can also include anypolynucleotide having at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more) sequence identity to a polynucleotide having the nucleicacid sequence of SEQ ID NO: 20, 21, 36, or 37.

By “vector” is meant a DNA construct that includes one or morepolynucleotides, or fragments thereof, encoding an sFGFR3 polypeptide(e.g., an sFGFR3 polypeptide or variant thereof, such as a polypeptidehaving the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3polypeptide including a signal peptide, such as a polypeptide having theamino acid sequence of SEQ ID NO: 18 or 34). The vector can be used toinfect a cell (e.g., a host cell or a cell of a patient having a humanskeletal growth retardation disorder, such as achondroplasia), whichresults in the translation of the polynucleotides of the vector into asFGFR3 polypeptide. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids.

The term “unit dosage form(s)” refers to physically discrete unit(s)suitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with anysuitable pharmaceutical excipient, carrier, or diluent.

The recitation herein of numerical ranges by endpoints is intended toinclude all numbers subsumed within that range (e.g., a recitation of 1to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

Other features and advantages of the invention will be apparent from thefollowing Detailed Description and from the claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1D are graphs showing sensorgrams of the sFGFR3 polypeptides.Sensorgrams are shown for sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10; FIG. 1A); sFGFR3_Del1(SEQ ID NO: 7) and sFGFR3 Del1-D3 (SEQ ID NO: 9; FIG. 1B);sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10), sFGFR3_Del4-LK1-LK2-C253S (SEQ IDNO: 11), and sFGFR3_Del4-LK1-LK2-D3 (SEQ ID NO: 12; FIG. 10); andsFGFR3_Del4 (SEQ ID NO: 1), sFGFR3_Del4-0253S (SEQ ID NO: 2), andsFGFR3_Del4-D3 (SEQ ID NO: 33; FIG. 1D).

FIGS. 2A-2C are images of Western blots of the sFGFR3 polypeptides.Western blots under reducing (R) and non-reducing (NR) conditions areshown for sFGFR3 Del1, sFGFR3 Del1-C253S (SEQ ID NO: 8), and sFGFR3Del1-D3 (FIG. 2A); sFGFR3_Del4-LK1-LK2, sFGFR3_Del4-LK1-LK2-C253S, andsFGFR3_Del4-LK1-LK2-D3 (FIG. 2B); and sFGFR3_Del4, sFGFR3_Del4-C253S,and sFGFR3_Del4-D3 (FIG. 2C).

FIGS. 3A-3B are graphs showing a sensorgram (FIG. 3A) and proliferationassays of sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 (FIG. 3B)using Fgfr3^(ach/+) chondrocyte cells in the presence of FGF2.

FIG. 4 is a graph showing luciferase signaling in Serum ResponseElement-Luciferase (SRE-Luc) HEK cells expressing FGFR3^(G380R)incubated with sFGFR3_Del4-D3 at 0 nM, 70 nM, and 280 nM with or without1 ng/mL of hFGF2 (* indicates p value <0.05; *** indicates a p value<0.001 compared to sFGFR3_Del4-D3 at 0 nM).

FIG. 5 is a graph showing the percentage of living animals (wild type(wt) and Fgfr3^(ach/+) mice) after 3 days of treatment with a low dose(0.25 mg/kg) of sFGFR3_Del4-D3 relative to age (days). The percentage ofliving wt mice receiving vehicle (PBS) is also shown.

FIG. 6 is an image showing the amino acid residues corresponding to theIg-like C2-type domains 1 (Ig1), 2 (Ig11), and 3 (Ig111) of wildtypeFGFR3 polypeptide (SEQ ID NO: 5 or 32), sFGFR3_Del4-C253S (SEQ ID NO:2), and a variant of sFGFR3_Del4-D3 (SEQ ID NO:33). sFGFR3_Del4-C253Sincludes an amino acid substitution of a cysteine residue with a serineresidue at position 253 of SEQ ID NO: 1.

FIGS. 7A-7B are images of Western blots of the sFGFR3 polypeptides.Western blots under reducing (R) and non-reducing (NR) conditions areshown for 2.3 mg/ml and 23 mg/ml sFGFR3 Del1-D3 (FIG. 7A) and 1.5 mg/mland 15 mg/ml sFGFR3 Del1-C253S (FIG. 7B).

FIGS. 8A-8B are graphs showing the melting temperature (T_(m)) ofsFGFR3_Del4-C253S in 20 mM phosphate, 40 mM NaCl, pH 7.5 buffer and 40mM citrate, 40 mM NaCl, pH 6.5 buffer (FIG. 8A) and the T_(m) ofsFGFR3_Del4-D3 in 20 mM phosphate, 40 mM NaCl, pH 7.5 buffer and 20 mMcitrate, 40 mM NaCl, pH 6.5 buffer (FIG. 8B).

FIGS. 9A-9G are graphs showing the fast protein liquid chromatography(FPLC) elution profiles of sFGFR3_Del4-D3. FIG. 9A: FPLC elutionprofiles are shown for sFGFR3_Del4-D3 at 0 minutes, 2 hours, and 24hours in cpm/fraction; FIGS. 9B-9D: sFGFR3_Del4-D3 administered byintravenous bolus at 1 minute, 15 minutes, 30 minute, 2 hours, and 24hours in cpm/fraction and as normalized to the highest peak; FIGS.9E-9G: sFGFR3_Del4-D3 administered by subcutaneous injection at 30minutes, 2 hours, 4 hours, and 24 hours in cpm/fraction and asnormalized to the highest peak (shown in FIG. 9C cont.).

FIGS. 10A-10B are graphs showing the percentage (%) of proliferation ofFgfr3^(ach/+) chondrocyte cells in the presence of the sFGFR3polypeptides. Fgfr3^(ach/+) chondrocyte proliferation is shown for 1ug/ml, 10 ug/ml, and 50 ug/ml of sFGFR3_Del4-D3 (FIG. 10A) and for 1ug/ml, 10 ug/ml, and 50 ug/ml of sFGFR3_Del4-C253S (FIG. 10B).

FIG. 11 is a graph showing the PK profiles of 2.5 mg/kg sFGFR3_Del4-D3administered subcutaneously and 2.5 mg/kg sFGFR3_Del4-D3 administeredintravenously.

FIG. 12 is a graph showing the concentration of ¹²⁵I-sFGFR3_Del4-D3 inkidney, liver, spleen, lung, and heart tissue at 30 minutes, 120minutes, and 1440 minutes after intravenous administration. Theconcentration is expressed as the percent of injected dose per gram (%ID/g).

FIG. 13 is a graph showing the concentration of ¹²⁵I-sFGFR3_Del4-D3 inkidney, liver, spleen, lung, and heart tissue at 30 minutes, 120minutes, 240 minutes, 480 minutes, and 1440 minutes after subcutaneousadministration. The concentration is expressed as % ID/g.

FIG. 14A-14B are graphs showing the concentration (c) and volume ofdistribution (V_(d)) of ¹²⁵I-sFGFR3_Del4-D3 in brain tissue. Shown isthe c of ¹²⁵I-sFGFR3_Del4-D3 before and after correction for vascularcontent and degradation at 30 minutes, 2 hours, and 24 hours afterintravenous bolus (FIG. 14A) and the V_(d) of ¹²⁵I-sFGFR3_Del4-D3 andRSA at 30 minutes, 2 hours, and 24 hours after intravenous bolus (FIG.14B).

FIG. 15 is a graph showing the percentage of surviving Fgfr3^(ach/+)mice administered sFGFR3_Del4-D3. Shown are the surviving wild typemice, Fgfr3^(ach/+) mice administered PBS as vehicle, Fgfr3^(ach/+) miceadministered 2.5 mg/kg sFGFR3_Del4-D3 once weekly, Fgfr3^(ach/+) miceadministered 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and Fgfr3^(ach/+)mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly over 22 days.

FIG. 16 is a graph showing the percentage (%) of locomotor and abdominalbreathing complications in Fgfr3^(ach/+) mice administered PBS asvehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly.

FIGS. 17A-170 are graphs and an x-ray radiograph showing the length ofFgfr3^(ach/+) mice administered sFGFR3_Del4-D3. Shown are the axiallength (FIG. 17A), tail length (FIG. 17B), and tibia length (FIG. 17C)of wild type mice administered PBS as vehicle, and Fgfr3^(ach/+) miceadministered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twiceweekly. Also shown is the x-ray radiograph (FIG. 17D) of wild type miceadministered PBS as vehicle and Fgfr3^(ach/+) mice administered PBS asvehicle, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kgsFGFR3_Del4-D3 twice weekly. All measurements are in millimeters (mm).

FIGS. 18A-18B are a graph showing the cranium ratio and an x-rayradiograph showing the skulls of Fgfr3^(ach/+) mice administeredsFGFR3_Del4-D3, respectively. Shown in the graph (FIG. 18A) is thecranium ratio (L/VV) of wild type mice administered PBS as vehicle andFgfr3^(ach/+) mice administered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kgsFGFR3_Del4-D3 twice weekly. Shown in the x-ray radiograph (FIG. 18B) isthe skulls of wild type mice administered PBS as vehicle, Fgfr3^(ach/+)mice administered PBS as vehicle, wild type mice administered 10 mg/kgsFGFR3_Del4-D3 twice weekly, and Fgfr3^(ach/+) mice administered 10mg/kg sFGFR3_Del4-D3 twice weekly.

FIGS. 19A-19F are graphs showing the kinetic profile for the binding ofdifferent concentrations of hFGF1, FGF2, hFGF9, hFGF18, hFGF19, andhFGF21 to immobilized SFGFR3_DEL4-D3 in real time. Shown are the kineticprofiles for binding of hFGF1 at concentrations of 0.5 nM to 12 nM toimmobilized SFGFR3_DEL4-D3 (FIG. 19A); hFGF2 at concentrations of 2 nMto 10 nM to immobilized SFGFR3_DEL4-D3 (FIG. 19B); hFGF9 atconcentrations of 1 nM to 5 nM to immobilized SFGFR3_DEL4-D3 (FIG. 19C);hFGF18 at concentrations of 1 nM to 10 nM to immobilized SFGFR3_DEL4-D3(FIG. 19D); hFGF19 at concentrations of 2 nM to 20 nM to immobilizedSFGFR3_DEL4-D3 (FIG. 19E); and hFGF21 at concentrations of 100 nM to10000 nM to immobilized SFGFR3_DEL4-D3 (FIG. 19F). The darker,overlapping lines represent the 2:1 binding model used to generate theK_(d) values.

FIG. 20 is an image of a Western blot of non-induced wild type ATDC5 andretrovirally infected ATDC5 cells expressing FGFR3^(G380R).

FIG. 21 is a graph showing the induction of proliferation of ATDC5FGFR3^(G380R) cells in the presence of SFGFR3_DEL4-D3 for threeexperiments. Untreated ATDC5 FGFR3^(G380R) cells were used as a control.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that soluble fibroblast growth factor receptor 3(sFGFR3) polypeptides and variants thereof can be used to treat skeletalgrowth retardation disorders, such as achondroplasia, in a patient(e.g., a human, particularly an infant, a child, or an adolescent). Inparticular, sFGFR3 polypeptides of the invention feature a deletion of,e.g., amino acids 289 to 400 of SEQ ID NO: 5 or amino acids 311 to 422of SEQ ID NO: 32, to provide the following exemplary sFGFR3polypeptides: sFGFR3_Del4 including an amino acid substitution of acysteine residue with a serine residue at position 253(sFGFR3_Del4-C253S; SEQ ID NO: 2) and sFGFR3_Del4 including an extendedIg-like C2-type domain 3 (sFGFR3_Del4-D3; SEQ ID NO: 33) and variantsthereof, such as a sFGFR3 polypeptide having the amino acid sequence ofSEQ ID NO: 4. Additionally, the sFGFR3 polypeptides may include a signalpeptide, such as a sFGFR3 polypeptide having the amino acid sequence ofSEQ ID NO: 18 or 34. See U.S. Provisional Application No. 62/276,222 andInternational Application No. PCT/US16/12553 for a description ofsFGFR3_Del4 (SEQ ID NO: 1), each of which is hereby incorporated hereinby reference in their entirety.

For example, sFGFR3 polypeptides and variants thereof having at least85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) tothe amino acid sequence of SEQ ID NO: 1 can include an amino acidsubstitution that removes a cysteine residue at position 253 of SEQ IDNO: 1 (e.g. sFGFR3_Del4-C253S; a polypeptide having the amino acidsequence of SEQ ID NO: 2). In particular, an sFGFR3 polypeptide of theinvention can include a substitution of a cysteine residue at position253 of SEQ ID NO: 1 with, e.g., a serine residue. For example, thecysteine residue at position 253 is substituted with a serine residueor, e.g., another conservative amino acid substitution, such as alanine,glycine, proline, or threonine.

The sFGFR3 polypeptides can also include a polypeptide sequence havingat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity) amino acid sequence identity to aminoacid residues 23 to 357 of SEQ ID NO: 32, in which the polypeptide lacksa signal peptide and a transmembrane domain of FGFR3 and (i) is lessthan 500 amino acids in length; (ii) comprises 200 consecutive aminoacids or fewer of an intracellular domain of FGFR3; and/or (iii) lacks atyrosine kinase domain of FGFR3 (e.g., sFGFR3_Del4-D3; a polypeptidehaving the amino acid sequence of SEQ ID NO: 33). Methods foradministering the sFGFR3 polypeptides of the invention to treat skeletalgrowth retardation disorders (e.g., achondroplasia) in a patient (e.g.,a human, particularly an infant, a child, or an adolescent) are alsodescribed.

The sFGFR3 polypeptides, methods of production, methods of treatment,compositions, and kits of the invention are described herein.

Soluble Fibroblast Growth Factor Receptor 3 (sFGFR3) Polypeptides

The invention features sFGFR3 polypeptides and variants thereofformulated for the treatment of skeletal growth retardation disorders(e.g., achondroplasia). In particular, the sFGFR3 polypeptides can haveat least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to theamino acid sequence of SEQ ID NO: 1, in which the sFGFR3 polypeptideincludes an amino acid substitution that removes a cysteine residue atposition 253 of SEQ ID NO: 1 (e.g. sFGFR3_Del4-C253S; a polypeptidehaving the amino acid sequence of SEQ ID NO: 2). For example, thecysteine residue at position 253 of SEQ ID NO: 1 is substituted with aserine residue or a conservative amino acid substitution, such asalanine, glycine, proline, or threonine.

The sFGFR3 polypeptides and variants thereof can also include fragmentsof the amino acid sequence of SEQ ID NO: 2 (e.g., at least amino acids 1to 200, 1 to 205, 1 to 210, 1 to 215, 1 to 220, 1 to 225, 1 to 235, 1 to230, 1 to 240, 1 to 245, 1 to 250, 1 to 253, 1 to 255, 1 to 260, 1 to265, 1 to 275, 1 to 280, 1 to 285, 1 to 290, or 1 to 300, of SEQ ID NO:2) having at least 50% sequence identity (e.g., 55%, 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity) to SEQ ID NO: 2. Additionally,sFGFR3 polypeptides can include amino acids 1 to 301 of SEQ ID NO: 1, inwhich the sFGFR3 polypeptide includes an amino acid substitution of acysteine residue with a serine residue at position 253 of SEQ ID NO: 1(e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2).

The sFGFR3 polypeptides and variants thereof can also include fragmentsof the amino acid sequence of SEQ ID NO: 33 (e.g., at least amino acids1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, 1 to 250, 1 to 260, 1to 270, 1 to 280, 1 to 290, 1 to 300, 1 to 310, 1 to 320, 1 to 330, 1 to340, 1 to 340, or 1 to 345 of SEQ ID NO: 33) having at least 50%sequence identity (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity) to SEQ ID NO: 33. In addition, the cysteine residueat position 253 of SEQ ID NO: 4 or 33 and/or position 316 of SEQ ID NO:4, if present, can be substituted with a serine residue or aconservative amino acid substitution, such as alanine, glycine, proline,or threonine.

Given the results described herein, the invention is not limited to aparticular sFGFR3 polypeptide or variants thereof. In addition to theexemplary sFGFR3 polypeptides and variants thereof discussed above, anypolypeptide that binds one or more FGFs (e.g., FGF1 (e.g., a polypeptidehaving the amino acid sequence of SEQ ID NO: 13), FGF2 (e.g., apolypeptide having the amino acid sequence of SEQ ID NO: 14), FGF9(e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 15),FGF18 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:16), FGF19 (e.g., a polypeptide having the amino acid sequence of SEQ IDNO: 38), and/or FGF21 (e.g., a polypeptide having the amino acidsequence of SEQ ID NO: 39)) with similar binding affinity as the sFGFR3polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) canbe used in the methods, such as for treating a skeletal growthretardation disorder, e.g., achondroplasia. The sFGFR3 polypeptides canbe, for example, fragments of FGFR3 isoform 2 lacking exons 8 and 9encoding the C-terminal half of the Ig-like C2-type domain 3 and exon 10including the transmembrane domain (e.g., fragments of the amino acidsequence of SEQ ID NO: 5 or 32), corresponding to fragments of FGFR3transcript variant 2 (Accession No. NM_022965).

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4)) can include a signal peptide at the N-terminal position. Anexemplary signal peptide can include, but is not limited to, amino acids1 to 22 of SEQ ID NO: 6 (e.g., MGAPACALALCVAVAIVAGASS) or amino acids 1to 19 of SEQ ID NO: 35 (e.g., MMSFVSLLLVGILFHATQA). Accordingly, thesFGFR3 polypeptides include both secreted forms, which lack theN-terminal signal peptide, and non-secreted forms, which include theN-terminal signal peptide. For instance, a secreted sFGFR3 polypeptidecan include the amino acid sequence of SEQ ID NOs: 2, 4, or 33.Alternatively, the sFGFR3 polypeptide does include a signal peptide,such the amino acid sequence of SEQ ID NOs: 18, 19, or 34. One skilledin the art will appreciate that the position of the N-terminal signalpeptide will vary in different sFGFR3 polypeptides and can include, forexample, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 27, 30, or more amino acid residues on the N-terminus ofthe polypeptide. One of skill in the art can predict the position of asignal sequence cleavage site, e.g., by an appropriate computeralgorithm such as that described in Bendtsen et al. (J. Mol. Biol.340(4):783-795, 2004) and available on the Web atcbs.dtu.dk/services/SignalP/.

Additionally, sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO:2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4)or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) of the invention can be glycosylated. In particular, a sFGFR3polypeptide can be altered to increase or decrease the extent to whichthe sFGFR3 polypeptide is glycosylated. Addition or deletion ofglycosylation sites to an sFGFR3 polypeptide can be accomplished byaltering the amino acid sequence such that one or more glycosylationsites is created or removed. For example, N-linked glycosylation, inwhich an oligosaccharide is attached to the amide nitrogen of anasparagine residue, can occur at position Asn76, Asn148, Asn169, Asn203, Asn240, Asn272, and/or Asn 294 of the amino acid sequence ofsFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 4 or 33),and variants thereof. One or more of these Asn residues can also besubstituted to remove the glycosylation site. For instance, O-linkedglycosylation, in which an oligosaccharide is attached to an oxygen atomof an amino acid residue, can occur at position Ser109, Thr126, Ser199,Ser274, Thr281, Ser298, Ser299, and/or Thr301 of the amino acid sequenceof sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33),variants thereof (SEQ ID NO: 4), and sFGFR3 polypeptides including asignal peptide (SEQ ID NO: 18 or 34). Additionally, O-linkedglycosylation can occur at position Ser310 and/or Ser321 ofsFGFR3_Del4-D3 (SEQ ID NO: 33) and variants thereof (SEQ ID NO: 4). Oneor more of these Ser or Thr residues can also be substituted to removethe glycosylation site.

sFGFR3 Fusion Polypeptides

sFGFR3 polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO:2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4)or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be fused to a functional domain from a heterologous polypeptide(e.g., a fragment crystallizable region of an immunoglobulin (Fc region;such as a polypeptide having the amino acid sequence of SEQ ID NOs: 25and 26) or human serum albumin (HSA; such as a polypeptide having theamino acid sequence of SEQ ID NO: 27)) to provide a sFGFR3 fusionpolypeptide. Optionally, a flexible linker, can be included between thesFGFR3 polypeptide and the heterologous polypeptide (e.g., an Fc regionor HSA), such as a serine or glycine-rich sequence (e.g., a poly-glycineor a poly-glycine/serine linker, such as SEQ ID NOs: 28 and 29).

For example, the sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO:2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4)or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be a fusion polypeptide including, e.g., an Fc region of animmunoglobulin at the N-terminal or C-terminal domain. In particular,useful Fc regions can include the Fc fragment of any immunoglobulinmolecule, including IgG, IgM, IgA, IgD, or IgE and their varioussubclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2) from anymammal (e.g., a human). For instance, the Fc fragment human IgG-1 (SEQID NO: 25) or a variant of human IgG-1, such as a variant including asubstitution of asparagine at position 297 of SEQ ID NO: 25 with alanine(e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 26).The Fc fragments of the invention can include, for example, the CH2 andCH3 domains of the heavy chain and any portion of the hinge region. ThesFGFR3 fusion polypeptides of the invention can also include, e.g., amonomeric Fc, such as a CH2 or CH3 domain. The Fc region may optionallybe glycosylated at any appropriate one or more amino acid residues knownto those skilled in the art. An Fc fragment as described herein may have1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 50, or more additions, deletions, or substitutionsrelative to any of the Fc fragments described herein.

Additionally, the sFGFR3 polypeptides (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be conjugated to other molecules at the N-terminal orC-terminal domain for the purpose of improving the solubility andstability of the protein in aqueous solution. Examples of such moleculesinclude human serum albumin (HSA), PEG, PSA, and bovine serum albumin(BSA). For instance, the sFGFR3 polypeptides can be conjugated to humanHSA (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:27) or a fragment thereof.

The sFGFR3 fusion polypeptides can include a peptide linker regionbetween the sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) andthe heterologous polypeptide (e.g., an Fc region or HSA). The linkerregion may be of any sequence and length that allows the sFGFR3 toremain biologically active, e.g., not sterically hindered. Exemplarylinker lengths are between 1 and 200 amino acid residues, e.g., 1-5,6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55,56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-110,111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190,or 191-200 amino acid residues. For instance, linkers include or consistof flexible portions, e.g., regions without significant fixed secondaryor tertiary structure. Preferred ranges are 5 to 25 and 10 to 20 aminoacids in length. Such flexibility is generally increased if the aminoacids are small and do not have bulky side chains that impede rotationor bending of the amino acid chain. Thus, preferably the peptide linkerof the present invention has an increased content of small amino acids,in particular of glycines, alanines, serines, threonines, leucines andisoleucines.

Exemplary flexible linkers are glycine-rich linkers, e.g., containing atleast 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycineresidues. Linkers may also contain, e.g., serine-rich linkers, e.g.,containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even100% serine residues. In some cases, the amino acid sequence of a linkerconsists only of glycine and serine residues. For example, the linkercan be the amino acid sequence of GGGGAGGGG (SEQ ID NO: 28) orGGGGSGGGGSGGGGS (SEQ ID NO: 29). A linker can optionally be glycosylatedat any appropriate one or more amino acid residues. The linker can alsobe absent, in which the FGFR3 polypeptide and the heterologouspolypeptide (e.g., an Fc region or HSA) are fused together directly,with no intervening residues.

Polynucleotides Encoding the sFGFR3 Polypeptides

The invention further includes polynucleotides encoding the sFGFR3polypeptides (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptideincluding a signal peptide (SEQ ID NO: 18 or 34)) that can be used totreat skeletal growth retardation disorders (e.g., achondroplasia) in apatient (e.g., a human, such as an infant, a child, or an adolescent),such as SEQ ID NOs: 20, 21, 36, or 37. For example, the polynucleotidecan be the nucleic acid sequence of SEQ ID NO: 20 or 36, which encodesFGFR3_Del4-C253S (SEQ ID NO: 2), or a variant having at least 85%sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acidsequence of SEQ ID NO: 20 or 36. Additionally, the polynucleotide can bethe nucleic acid sequence of SEQ ID NO: 21 or 37, which encodessFGFR3_Del4-D3 (SEQ ID NO: 33), having at least 85% sequence identity(e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity) to the nucleic acid sequence of SEQ IDNO: 21 or 37. The invention also includes polynucleotides encodingsFGFR3 fusion polypeptides (e.g., a sFGFR3 polypeptide fused to aheterologous polypeptide, such as a Fc region or HSA) andpolynucleotides encoding sFGFR3 polypeptides without a signal peptide(e.g., polypeptides having the amino acid sequence of SEQ ID NOs: 2, 4,and 33) or with a signal peptide (e.g., polypeptides having the aminoacid sequence of SEQ ID NOs: 18, 19, and 34). Additionally, theinvention includes polynucleotides include one or more mutations toalter any of the glycosylation sites described herein.

Optionally, the sFGFR3 polynucleotides of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can be codon optimized to alterthe codons in the nucleic acid, in particular to reflect the typicalcodon usage of the host organism (e.g., a human) without altering thesFGFR3 polypeptide encoded by the nucleic acid sequence of thepolynucleotide. Codon-optimized polynucleotides (e.g., a polynucleotidehaving the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37) can,e.g., facilitate genetic manipulations by decreasing the GC contentand/or for expression in a host cell (e.g., a HEK 293 cell or a CHOcell). Codon-optimization can be performed by the skilled person, e.g.by using online tools such as the JAVA Codon Adaption Tool (www.jcat.de)or Integrated DNA Technologies Tool (www.eu.idtdna.com/CodonOpt) bysimply entering the nucleic acid sequence of the polynucleotide and thehost organism for which the codons are to be optimized. The codon usageof different organisms is available in online databases, for example,www.kazusa.or.jp/codon.

Host Cells for Expression of the sFGFR3 Polypeptides

Mammalian cells can be used as host cells for expression of the sFGFR3polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)).Exemplary mammalian cell types useful in the methods include, but arenot limited to, human embryonic kidney (HEK; e.g., HEK 293) cells,Chinese Hamster Ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHKcells, COS-7 cells, HeLa cells, PC3 cells, Vero cells, MC3T3 cells, NSOcells, Sp2/0 cells, VERY cells, BHK, MDCK cells, W138 cells, BT483cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, NSO cells,CRL7O3O cells, and HsS78Bst cells, or any other suitable mammalian hostcell known in the art. Alternatively, E. coli cells can be used as hostcells for expression of the sFGFR3 polypeptides. Examples of E. colistrains include, but are not limited to, E. coli 294 (ATCC®31,446), E.coli A 1776 (ATCC®31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), E. coliRV308 (ATCC®31,608), or any other suitable E. coli strain known in theart.

Vectors Including Polynucleotides Encoding the sFGFR3 Polypeptides

The invention also features recombinant vectors including any one ormore of the polynucleotides described above. The vectors of theinvention can be used to deliver a polynucleotide encoding a sFGFR3polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)),which can include mammalian, viral, and bacterial expression vectors.For example, the vectors can be plasmids, artificial chromosomes (e.g.BAG, PAC, and YAC), and virus or phage vectors, and may optionallyinclude a promoter, enhancer, or regulator for the expression of thepolynucleotide. The vectors can also contain one or more selectablemarker genes, such as an ampicillin, neomycin, and/or kanamycinresistance gene in the case of a bacterial plasmid or a resistance genefor a fungal vector. Vectors can be used in vitro for the production ofDNA or RNA or used to transfect or transform a host cell, such as amammalian host cell for the production of a sFGFR3 polypeptide encodedby the vector. The vectors can also be adapted to be used in vivo in amethod of gene therapy.

Exemplary viral vectors that can be used to deliver a polynucleotideencoding a sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S(SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof(SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQID NO: 18 or 34)) include a retrovirus, adenovirus (e.g., Ad2, Ad5,Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, and Pan9(also known as AdC68)), parvovirus (e.g., adeno-associated viruses),coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g.,influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitisvirus), paramyxovirus (e.g. measles and Sendai), positive strand RNAviruses, such as picornavirus and alphavirus, and double stranded DNAviruses including adenovirus, herpesvirus (e.g., Herpes Simplex virustypes 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g.,vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Otherviruses useful for delivering polynucleotides encoding sFGFR3polypeptides include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus. Examples of retrovirusesinclude avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-typeviruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J. M.,Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996).

Methods of Production

Polynucleotides encoding sFGFR3 polypeptides of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can be produced by any methodknown in the art. For instance, a polynucleotide is generated usingmolecular cloning methods and is placed within a vector, such as aplasmid, an artificial chromosome, a viral vector, or a phage vector.The vector is used to transform the polynucleotide into a host cellappropriate for the expression of the sFGFR3 polypeptide.

Nucleic Acid Vector Construction and Host Cells

The sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be produced from a host cell. The polynucleotides (e.g.,polynucleotides having the nucleic acid sequence of SEQ ID NO: 20, 21,36, or 37 and variants thereof) encoding sFGFR3 polypeptides can beincluded in vectors that can be introduced into the host cell byconventional techniques known in the art (e.g., transformation,transfection, electroporation, calcium phosphate precipitation, directmicroinjection, or infection). The choice of vector depends in part onthe host cells to be used. Generally, host cells are of eitherprokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

A polynucleotide encoding an sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can be prepared by a variety ofmethods known in the art. These methods include, but are not limited to,oligonucleotide-mediated (or site-directed) mutagenesis and PCRmutagenesis. A polynucleotide encoding an sFGFR3 polypeptide can beobtained using standard techniques, e.g., gene synthesis. Alternatively,a polynucleotide encoding a wild-type sFGFR3 polypeptide (e.g., apolypeptide having the amino acid sequence of SEQ ID NO: 5 or 32) can bemutated to contain specific amino acid substitutions (e.g., an aminoacid substitution of a cysteine residue with a serine residue or aconservative amino acid substitution, such as alanine, glycine, proline,or threonine, at position 253 of SEQ ID NO: 33 and/or position 316 ofSEQ ID NO: 4) using standard techniques in the art, e.g., QuikChange™mutagenesis. Polynucleotides encoding an sFGFR3 polypeptide can besynthesized using, e.g., a nucleotide synthesizer or PCR techniques.

Polynucleotides encoding sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can be inserted into a vectorcapable of replicating and expressing the polynucleotide in prokaryoticor eukaryotic host cells. Exemplary vectors useful in the methods caninclude, but are not limited to, a plasmid, an artificial chromosome, aviral vector, and a phage vector. For example, a viral vector caninclude the viral vectors described above, such as a retroviral vector,adenoviral vector, or poxviral vector (e.g., vaccinia viral vector, suchas Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, andalphaviral vector)) containing the nucleic acid sequence of apolynucleotide encoding the sFGFR3 polypeptide. Each vector can containvarious components that may be adjusted and optimized for compatibilitywith the particular host cell. For example, the vector components mayinclude, but are not limited to, an origin of replication, a selectionmarker gene, a promoter, a ribosome binding site, a signal sequence, thenucleic acid sequence of the polynucleotide encoding the sFGFR3polypeptide, and/or a transcription termination sequence.

The above-described vectors may be introduced into appropriate hostcells (e.g., HEK 293 cells or CHO cells) using conventional techniquesin the art, e.g., transformation, transfection, electroporation, calciumphosphate precipitation, and direct microinjection. Once the vectors areintroduced into host cells for the production of an sFGFR3 polypeptideof the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3(SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3polypeptide including a signal peptide (SEQ ID NO: 18 or 34)), hostcells are cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the polynucleotides (e.g. SEQ ID NOs: 20 and 21 and variantsthereof) encoding the sFGFR3 polypeptide. Methods for expression oftherapeutic proteins, such as sFGFR3 polypeptides, are known in the art,see, for example, Paulina Balbas, Argelia Lorence (eds.) RecombinantGene Expression: Reviews and Protocols (Methods in Molecular Biology),Humana Press; 2nd ed. 2004 (Jul. 20, 2004) and Vladimir Voynov andJustin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols(Methods in Molecular Biology) Humana Press; 2nd ed. 2012 (Jun. 28,2012), each of which is hereby incorporated by reference in itsentirety.

sFGFR3 Polypeptide Production, Recovery, and Purification

Host cells (e.g., HEK 293 cells or CHO cells) used to produce the sFGFR3polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)) canbe grown in media known in the art and suitable for culturing of theselected host cells. Examples of suitable media for mammalian host cellsinclude Minimal Essential Medium (MEM), Dulbecco's Modified Eagle'sMedium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetalbovine serum (FBS), and RPMI-1640. Examples of suitable media forbacterial host cells include Luria broth (LB) plus necessarysupplements, such as a selection agent, e.g., ampicillin. Host cells arecultured at suitable temperatures, such as from about 20° C. to about39° C., e.g., from 25° C. to about 37° C., preferably 37° C., and CO₂levels, such as 5 to 10% (preferably 8%). The pH of the medium isgenerally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the hostorganism. If an inducible promoter is used in the expression vector,sFGFR3 polypeptide expression is induced under conditions suitable forthe activation of the promoter.

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be recovered from the supernatant of the host cell.Alternatively, the sFGFR3 polypeptide can be recovered by disrupting thehost cell (e.g., using osmotic shock, sonication, or lysis), followed bycentrifugation or filtration to remove the sFGFR3 polypeptide. Uponrecovery of the sFGFR3 polypeptide, the sFGFR3 polypeptide can then befurther purified. An sFGFR3 polypeptide can be purified by any methodknown in the art of protein purification, such as protein A affinity,other chromatography (e.g., ion exchange, affinity, and size-exclusioncolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins (seeProcess Scale Purification of Antibodies, Uwe Gottschalk (ed.) JohnWiley & Sons, Inc., 2009, hereby incorporated by reference in itsentirety).

Optionally, the sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can be conjugated to a detectablelabel for purification. Examples of suitable labels for use inpurification of the sFGFR3 polypeptides include, but are not limited to,a protein tag, a fluorophore, a chromophore, a radiolabel, a metalcolloid, an enzyme, or a chemiluminescent, or bioluminescent molecule.In particular, protein tags that are useful for purification of thesFGFR3 polypeptides can include, but are not limited to, chromatographytags (e.g., peptide tags consisting of polyanionic amino acids, such asa FLAG-tag, or a hemagglutinin “HA” tag), affinity tags (e.g., apoly(His) tag, chitin binding protein (CBP), maltose binding protein(MBP), or glutathione-S-transferase (GST)), solubilization tags (e.g.,thioredoxin (TRX) and poly(NANP)), epitope tags (e.g., V5-tag, Myc-tag,and HA-tag), or fluorescence tags (e.g., GFP, GFP variants, RFP, and RFPvariants).

Methods of Treatment

Provided herein are methods for treating a skeletal growth retardationdisorder in a patient, such as a patient having achondroplasia (e.g., ahuman having achondroplasia). In particular, the patient is one thatexhibits or is likely to develop one or more symptoms of a skeletalgrowth retardation disorder (e.g., achondroplasia). The method involvesadministering an sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) to the patient having a skeletalgrowth retardation disorder, such as a patient having achondroplasia(e.g., a human having achondroplasia). In particular, the methodinvolves administering sFGFR3_Del4-C253S (SEQ ID NO: 2) orsFGFR3_Del4-D3 (SEQ ID NO: 33) to the patient having a skeletal growthretardation disorder, such as a patient having achondroplasia (e.g., ahuman having achondroplasia). For example, the patient is an infant orchild having a skeletal growth retardation disorder, such as an infant,a child, or an adolescent having achondroplasia (e.g., a human havingachondroplasia).

The patient (e.g., a human) can be treated before symptoms of a skeletalgrowth retardation disorder (e.g., achondroplasia) appear or aftersymptoms of a skeletal growth retardation disorder (e.g.,achondroplasia) develop. In particular, patients that can be treatedwith a sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ IDNO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18or 34)) are those exhibiting symptoms including, but not limited to,short limbs, short trunk, bowlegs, a waddling gait, skull malformations,cloverleaf skull, craniosynostosis, wormian bones, anomalies of thehands, anomalies of the feet, hitchhiker thumb, and/or chest anomalies.Furthermore, treatment with an sFGFR3 polypeptide can result in animprovement in one or more of the aforementioned symptoms of a skeletalgrowth retardation disorder (e.g., relative to an untreated patient),such as achondroplasia.

The patient (e.g., a human) can be diagnosed with a skeletal growthretardation disorder, such as achondroplasia, before administration ofan sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)). Additionally, the patient having a skeletal growth retardationdisorder, such as achondroplasia, can be one that has not previouslybeen treated with an sFGFR3 polypeptide.

Skeletal Growth Retardation Disorders

Skeletal growth retardation disorders can be treated by administering ansFGFR3 polypeptide as described herein to a patient (e.g., a human) inneed thereof. The method involves administering to the patient (e.g., ahuman) having the skeletal growth retardation disorder an sFGFR3polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)).Skeletal growth retardation disorders that can be treated with thesFGFR3 polypeptides are characterized by deformities and/ormalformations of the bones and can include, but are not limited to,FGFR3-related skeletal diseases. In particular, the patient is treatedwith sFGFR3_Del4-C253S (SEQ ID NO: 2) or sFGFR3_Del4-D3 (SEQ ID NO: 33).

Administration of an sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can treat a skeletal growthretardation disorder including, but not limited to, achondroplasia,achondrogenesis, acrodysostosis, acromesomelic dysplasia,atelosteogenesis, camptomelic dysplasia, chondrodysplasia punctata,rhizomelic type of chondrodysplasia punctata, cleidocranial dysostosis,congenital short femur, Crouzon syndrome, Apert syndrome, Jackson-Weisssyndrome, Pfeiffer syndrome, Crouzonodermoskeletal syndrome, dactyly,brachydactyly, camptodactyly, polydactyly, syndactyly, diastrophicdysplasia, dwarfism, dyssegmental dysplasia, enchondromatosis,fibrochondrogenesis, fibrous dysplasia, hereditary multiple exostoses,hypophosphatasia, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome,Kniest dysplasia, Kniest syndrome, Langer-type mesomelic dysplasia,Marfan syndrome, McCune-Albright syndrome, micromelia, metaphysealdysplasia, Jansen-type metaphyseal dysplasia, metatrophic dysplasia,Morquio syndrome, Nievergelt-type mesomelic dysplasia, neurofibromatosis(such as type 1 (e.g., with bone manifestations or without bonemanifestations), type 2, or schwannomatosis), osteoarthritis,osteochondrodysplasia, osteogenesis imperfecta, perinatal lethal type ofosteogenesis imperfecta, osteopetrosis, osteopoikilosis, peripheraldysostosis, Reinhardt syndrome, Roberts syndrome, Robinow syndrome,short-rib polydactyly syndromes, short stature, spondyloepiphysealdysplasia congenita, and spondyloepimetaphyseal dysplasia.

For instance, the sFGFR3 polypeptides of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can be used to treat symptomsassociated with a skeletal growth retardation disorder, including thedisorders described above, such as achondroplasia. Non-limiting examplesof symptoms of skeletal growth retardation disorders that can be treatedwith the sFGFR3 polypeptides, include short limbs and trunk, bowlegs, awaddling gait, skull malformations (e.g., a large head), cloverleafskull, craniosynostosis (e.g., premature fusion of the bones in theskull), wormian bones (e.g., abnormal thread-like connections betweenthe bones in the skull), anomalies of the hands and feet (e.g.,polydactyly or extra fingers), “hitchhiker” thumbs and abnormalfingernails and toenails, and chest anomalies (e.g., pear-shaped chestor narrow thorax). Additional symptoms that can treated by administeringsFGFR3 polypeptides can also include non-skeletal abnormalities inpatients having skeletal growth retardation disorders, such as anomaliesof the eyes, mouth, and ears, such as congenital cataracts, myopia,cleft palate, or deafness; brain malformations, such as hydrocephaly,porencephaly, hydranencephaly, or agenesis of the corpus callosum; heartdefects, such as atrial septal defect, patent ductus arteriosus, ortransposition of the great vessels; developmental delays; or mentaldisabilities.

Treatment with the sFGFR3 polypeptides of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can also increase survival ofpatients (e.g., humans) with skeletal growth retardation disorders(e.g., achondroplasia). For example, the survival rate of patientstreated with the sFGFR3 polypeptides can increase by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more relative to, e.g., anuntreated patient with a skeletal growth retardation disorder (e.g.,achondroplasia), over a treatment period of days, months, years, orlonger. In particular, administration of sFGFR3_Del4-D3 can increasesurvival of patients (e.g., humans) with skeletal growth retardationdisorders (e.g., relative to an untreated patient), such asachondroplasia.

Any skeletal growth retardation disorder that is a FGFR3-relatedskeletal disease (e.g., caused by or associated with overactivation ofFGFR3 as result of a gain-of-function FGFR3 mutation) can be treated byadministering an sFGFR3 polypeptide of the invention ((e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) to a patient (e.g., a human). Forexample, FGFR3-related skeletal diseases can include, but are notlimited to, achondroplasia, thanatophoric dysplasia type I (TDI),thanatophoric dysplasia type II (TDII), severe achondroplasia withdevelopmental delay and acanthosis nigricans (SADDAN),hypochondroplasia, and craniosynostosis (e.g., Muenke syndrome, Crouzonsyndrome, and Crouzonodermoskeletal syndrome).

Patients (e.g., humans) with mutations in the FGFR3 gene associated withdifferent FGFR3-related skeletal disorders, such as achondroplasia,hypochondroplasia, SADDAN, TDI, and TDII, can be treated with sFGFR3polypeptides of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)).For example, the sFGFR3 polypeptides can be administered to treatachondroplasia resulting from the G380R, G375C, G346E or S279C mutationsof the FGFR3 gene. Administration of the sFGFR3 polypeptides can be usedto treat the following exemplary FGFR3-related skeletal disorders:hypochondroplasia resulting from the G375C, G346E or S279C mutations ofthe FGFR3 gene; TDI resulting from the R248C, S248C, G370C, S371C,Y373C, X807R, X807C, X807G, X8075, X807W and K650M mutations of theFGFR3 gene; TDII resulting from the K650E mutation of the FGFR3 gene;and SADDAN resulting from the K650M mutation of the FGFR3 gene.

Any of the aforementioned mutations in the FGFR3 gene (e.g., the G380Rmutation of the FGFR3 gene) can be detected in a sample from the patient(e.g., a human with achondroplasia, hypochondroplasia, SADDAN, TDI, andTDII) prior to or after treatment with an sFGFR3 polypeptide of theinvention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ IDNO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptideincluding a signal peptide (SEQ ID NO: 18 or 34)). Additionally, theparents of the patient and/or fetal samples (e.g., fetal nucleic acidobtained from maternal blood, placental, or fetal samples) can be testedby methods known in the art for the mutation in the FGFR3 gene todetermine their need for treatment.

Achondroplasia

Achondroplasia is the most common cause of dwarfism in humans and can betreated by administering sFGFR3 polypeptides as described herein. Inparticular, achondroplasia can be treated by administering an sFGFR3polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)).Accordingly, administration of the sFGFR3 polypeptides can result in animprovement in symptoms including, but not limited to, growthretardation, skull deformities, orthodontic defects, cervical cordcompression (with risk of death, e.g., from central apnea or seizures),spinal stenosis (e.g., leg and lower back pain), hydrocephalus (e.g.,requiring cerebral shunt surgery), hearing loss due to chronic otitis,cardiovascular disease, neurological disease, respiratory problems,fatigue, pain, numbness in the lower back and/or spine, and/or obesity.

Patients treated using the sFGFR3 polypeptides of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) can include infants, children, andadults with achondroplasia. In particular, infants are often diagnosedwith achondroplasia at birth, and thus, treatment with the sFGFR3polypeptides can begin as early as possible in the patient's life, e.g.,shortly after birth, or prior to birth (in utero).

Symptoms of achondroplasia in patients (e.g., humans) can also bemonitored prior to or after a patient is treated with an sFGFR3polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2),sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or asFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or 34)).For instance, symptoms of achondroplasia can be monitored prior totreatment to assess the severity of achondroplasia and condition of thepatient prior to performing the methods.

The methods can include diagnosis of achondroplasia in a patient andmonitoring the patient for changes in the symptoms of achondroplasia,such as changes in body weight and skull size (e.g., skull length and/orskull width) of the patient. Changes in body weight and skull size canbe monitored over a period of time, e.g., 1, 2, 3, 4 or more times permonth or per year or approximately every 1, 2, 3, 4, 5, 6, 7, 8, 12 or16 weeks over the course of treatment with the sFGFR3 polypeptide of theinvention (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ IDNO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptideincluding a signal peptide (SEQ ID NO: 18 or 34)). Body weight and/orskull size of the patient having achondroplasia can also be determinedat treatment specific events, such as before and/or after administrationof the sFGFR3 polypeptide.

For example, body weight and/or skull size can be measured in responseto administration of the sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)). Body weight can be measured byweighing the patient having achondroplasia on a scale, preferably in astandardized manner, such as with the same or no clothes or at a certaintime of the day, preferably in a fasting state (e.g., in the morningbefore breakfast or after at least 1, 2, 3, 4, 5 or more hours offasting). Skull size can be represented by length, height, width, and/orcircumference of the skull. Measurements can be performed using anyknown or self-devised standardized method. For a human subject, themeasurement of skull circumference is preferred, which can be measuredusing a flexible and non-stretchable material, such as a tape, wrappedaround the widest possible circumference of the head (e.g. from the mostprominent part of the forehead around to the widest part of the back ofthe head). The height of the skull of the subject (e.g., human) can alsobe determined from the underside of the chin to the uppermost point ofthe head. Preferably, any measurement is performed more than once, e.g.at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

Administration of sFGFR3 Polypeptides

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be administered by any route known in the art, such as byparenteral administration, enteral administration, or topicaladministration. In particular, the sFGFR3 polypeptide can beadministered to the patient having a skeletal growth retardationdisorder (e.g., achondroplasia) subcutaneously (e.g., by subcutaneousinjection), intravenously, intramuscularly, intra-arterially,intrathecally, or intraperitoneally.

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be administered to a patient (e.g., a human) at a predetermineddosage, such as in an effective amount to treat a skeletal growthretardation disorder (e.g., achondroplasia), without inducingsignificant toxicity. For example, sFGFR3 polypeptides can beadministered to a patient having skeletal growth retardation disorders(e.g., achondroplasia) in individual doses ranging from about 0.002mg/kg to about 50 mg/kg (e.g., from 2.5 mg/kg to 30 mg/kg, from 0.002mg/kg to 20 mg/kg, from 0.01 mg/kg to 2 mg/kg, from 0.2 mg/kg to 20mg/kg, from 0.01 mg/kg to 10 mg/kg, from 10 mg/kg to 100 mg/kg, from 0.1mg/kg to 50 mg/kg, 0.5 mg/kg to 20 mg/kg, 1.0 mg/kg to 10 mg/kg, 1.5mg/kg to 5 mg/kg, or 0.2 mg/kg to 3 mg/kg). In particular, the sFGFR3polypeptide can be administered in individual doses of, e.g., 0.001mg/kg to 50 mg/kg, such as 2.5 mg/kg to about 10 mg/kg.

Exemplary doses of an sFGFR3 polypeptide of the invention (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)) for administration to a patient(e.g., a human) having a skeletal growth retardation disorder (e.g.,achondroplasia) include, e.g., 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,or 50 mg/kg. These doses can be administered one or more times (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more times) per day, week, month,or year. For example, an sFGFR3 polypeptide can be administered topatients in a weekly dosage ranging, e.g., from about 0.0014 mg/kg/weekto about 140 mg/kg/week, e.g., about 0.14 mg/kg/week to about 105mg/kg/week, or, e.g., about 1.4 mg/kg/week to about 70 mg/kg/week (e.g.,2.5 mg/kg/week, 5 mg/kg/week, 10 mg/kg/week, 20 mg/kg/week, 30mg/kg/week, 40 mg/kg/week, or 50 mg/kg/week).

Gene Therapy

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can also be delivered through gene therapy, where a polynucleotideencoding the sFGFR3 polypeptide is delivered to tissues of interest andexpressed in vivo. Gene therapy methods are discussed, e.g., in Verme etal. (Nature 389: 239-242, 1997), Yamamoto et al. (Molecular Therapy 17:S67-S68, 2009), and Yamamoto et al., (J. Bone Miner. Res. 26: 135-142,2011), each of which is hereby incorporated by reference.

An sFGFR3 polypeptide of the invention (e.g. sFGFR3_Del4-C253S (SEQ IDNO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO:4) or a sFGFR3 polypeptide including a signal peptide (SEQ ID NO: 18 or34)) can be produced by the cells of a patient (e.g., a human) having askeletal growth retardation disorder (e.g., achondroplasia) byadministrating a vector (e.g., a plasmid, an artificial chromosome (e.g.BAG, PAC, and YAC), or a viral vector) containing the nucleic acidsequence of a polynucleotide encoding the sFGFR3 polypeptide. Forexample, a viral vector can be a retroviral vector, adenoviral vector,or poxviral vector (e.g., vaccinia viral vector, such as ModifiedVaccinia Ankara (MVA)), adeno-associated viral vector, or alphaviralvector. The vector, once inside a cell of the patient (e.g., a human)having a skeletal growth retardation disorder (e.g., achondroplasia),by, e.g., transformation, transfection, electroporation, calciumphosphate precipitation, or direct microinjection, will promoteexpression of the sFGFR3 polypeptide, which is then secreted from thecell. The invention further includes cell-based therapies, in which thepatient (e.g., a human) is administered a cell expressing the sFGFR3polypeptide.

Pharmaceutical Compositions

Pharmaceutical compositions of the invention can include an sFGFR3polypeptide (e.g. sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQID NO: 33), and variants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptideincluding a signal peptide (SEQ ID NO: 18 or 34)), polynucleotide,vector, and/or host cell of the invention. Compositions including ansFGFR3 polypeptide, polynucleotide, vector, and/or host cell can beformulated at a range of dosages, in a variety of formulations, and incombination with pharmaceutically acceptable excipients, carriers, ordiluents.

A pharmaceutical composition including an sFGFR3 polypeptide (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/orhost cell of the invention can be formulated at a specific dosage, suchas a dosage that is effective for treating a patient (e.g., a human)skeletal growth retardation disorder (e.g., achondroplasia), withoutinducing significant toxicity. For example, the compositions can beformulated to include between about 1 mg/mL and about 500 mg/mL of thesFGFR3 polypeptide (e.g., between 10 mg/mL and 300 mg/mL, 20 mg/mL and120 mg/mL, 40 mg/mL and 200 mg/mL, 30 mg/mL and 150 mg/mL, 40 mg/mL and100 mg/mL, 50 mg/mL and 80 mg/mL, or 60 mg/mL and 70 mg/mL of the sFGFR3polypeptide).

The pharmaceutical compositions including an sFGFR3 polypeptide (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/orhost cell of the invention can be prepared in a variety of forms, suchas a liquid solution, dispersion or suspension, powder, or other orderedstructure suitable for stable storage. For example, compositionsincluding an sFGFR3 polypeptide intended for systemic or local deliverycan be in the form of injectable or infusible solutions, such as forparenteral administration (e.g., subcutaneous, intravenous,intramuscular, intra-arterial, intrathecal, or intraperitonealadministration). sFGFR3 compositions for injection (e.g., subcutaneousor intravenous injection) can be formulated using a sterile solution orany pharmaceutically acceptable liquid as a vehicle. Pharmaceuticallyacceptable vehicles include, but are not limited to, sterile water,physiological saline, and cell culture media (e.g., Dulbecco's ModifiedEagle Medium (DMEM), α-Modified Eagles Medium (α-MEM), F-12 medium).Formulation methods are known in the art, see e.g., Banga (ed.)Therapeutic Peptides and Proteins: Formulation, Processing and DeliverySystems (2nd ed.) Taylor & Francis Group, CRC Press (2006), which ishereby incorporated by reference in its entirety.

Compositions including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S(SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof(SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQID NO: 18 or 34)), polynucleotide, vector, and/or host cell of theinvention can be provided to patients (e.g., humans) having skeletalgrowth retardation disorders (e.g. achondroplasia) in combination withpharmaceutically acceptable excipients, carriers, or diluents.Acceptable excipients, carriers, or diluents can include buffers,antioxidants, preservatives, polymers, amino acids, and carbohydrates.Aqueous excipients, carriers, or diluents can include water,water-alcohol solutions, emulsions or suspensions including saline,buffered medical parenteral vehicles including sodium chloride solution,Ringer's dextrose solution, dextrose plus sodium chloride solution,Ringer's solution containing lactose, and fixed oils. Examples ofnon-aqueous excipients, carriers, or diluents are propylene glycol,polyethylene glycol, vegetable oil, fish oil, and injectable organicesters.

Pharmaceutically acceptable salts can also be included in thecompositions including an sFGFR3 polypeptide (e.g. sFGFR3_Del4-C253S(SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof(SEQ ID NO: 4) or a sFGFR3 polypeptide including a signal peptide (SEQID NO: 18 or 34)), polynucleotide, vector, and/or host cell of theinvention. Exemplary pharmaceutically acceptable salts can includemineral acid salts (e.g., hydrochlorides, hydrobromides, phosphates, andsulfates) and salts of organic acids (e.g., acetates, propionates,malonates, and benzoates). Additionally, auxiliary substances, such aswetting or emulsifying agents and pH buffering substances, can bepresent. A thorough discussion of pharmaceutically acceptableexcipients, carriers, and diluents is available in Remington: TheScience and Practice of Pharmacy, 22^(nd) Ed., Allen (2012), which ishereby incorporated by reference in its entirety.

Pharmaceutical compositions including an sFGFR3 polypeptide (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)), polynucleotide, vector, and/orhost cell of the invention can also be formulated with a carrier thatwill protect the sFGFR3 polypeptide against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. For example, the sFGFR3 composition can be entrappedin microcapsules prepared by coacervation techniques or by interfacialpolymerization, such as hydroxymethylcellulose, gelatin, orpoly-(methylmethacylate) microcapsules; colloidal drug delivery systems(e.g., liposomes, albumin microspheres, microemulsions, nano-particles,or nanocapsules); or macroemulsions. Additionally, an sFGFR3 compositioncan be formulated as a sustained-release composition. For example,sustained-release compositions can include semi-permeable matrices ofsolid hydrophobic polymers containing the sFGFR3 polypeptides,polynucleotides, vectors, or host cells of the invention, in which thematrices are in the form of shaped articles, such as films ormicrocapsules.

Kits

Kits of the invention can include one or more sFGFR3 polypeptides (e.g.sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), andvariants thereof (SEQ ID NO: 4) or a sFGFR3 polypeptide including asignal peptide (SEQ ID NO: 18 or 34)), polynucleotides, vectors, and/orcells of the invention as described herein. For example, the sFGFR3polypeptide, polynucleotide, vector, and/or cell can be present in acontainer (e.g., a glass vial) in liquid form (e.g., in water or abuffered salt solution, such as, 2 mM to 20 mM of sodium phosphate, pH6.5 or 7.0, and 25 mM to 250 mM sodium chloride). Alternatively, thesFGFR3 polypeptide, polynucleotide, and/or vector is present in acontainer (e.g., a glass vial) in lyophilized form, which can optionallyinclude a diluent (e.g., water or a buffered salt solution) forreconstitution of the lyophilized sFGFR3 polypeptide, polynucleotide,vector, and/or cell into liquid form prior to administration. The sFGFR3polypeptide, polynucleotide, vector, and/or cell can also be present ina kit in another formulation as described herein. The kit components canbe provided in dosage form to facilitate administration, and optionally,can include materials required for administration and/or instructionsfor patient treatment consistent with the methods. For example, the kitcan include instructions for use, which guides the user (e.g., thephysician) with respect to the administration of the sFGFR3 polypeptide,polynucleotide, vector, and/or cell.

EXAMPLES

The following examples are intended to illustrate, rather than limit,the disclosure. These studies feature the administration of the sFGFR3polypeptides of sFGFR3_Del4-C253S (SEQ ID NO: 2) and sFGFR3_Del4-D3 (SEQID NO: 33) to patients (e.g., humans) having achondroplasia, to treatachondroplasia and symptoms associated therewith.

Example 1: Production of sFGFR3 Polypeptides

sFGFR3_Del4-C253S (SEQ ID NO: 2) and sFGFR3_Del4-D3 (SEQ ID NO: 33) wereproduced by transient transfection in three different suspension celltypes: HEK 293 freestyle, CHO—S freestyle cells and Expi CHO—S cells.For production in HEK 293 freestyle and CHO—S freestyle cells,transfection was performed using polyethylenimine(PEIpro®—Polyplus-transfection), according to the manufacturer'sdirections. Proteins were harvested after three days. For sFGFR3polypeptide production in Expi CHO—S cells, transfection was performedusing Expifectamine as described by the manufacturer using the HighTiter production protocol. A time course was performed and sFGFR3polypeptides were optimally harvested after 12 days. Western blots werethen performed using 50 ng of sFGFR3 polypeptide. Classical western blotprotocols were used with B9 as a primary antibody (anti FGFR3, sc-13121,Santa Cruz) diluted 1:2000 in blocking buffer and anti-mouse IgGsecondary antibody (Anti-mouse IgG, #7076, Cell signaling) diluted1:5000 in blocking buffer.

Example 2: Purification of sFGFR3 Polypeptides

sFGFR3_Del4-C253S and sFGFR3_Del4-D3 were each purified using a two-steppurification process including ion exchange chromatography and sizeexclusion chromatography.

For ion exchange chromatography, 300 mL of culture supernatant waspurified by cross flow filtration (ÄKTA™ flux, GE Healthcare) using 5 μmand 0.2 μm capsules (KGF-A0504 TT and KMP-HEC 9204 TT, GE Healthcare,respectively). The purified sample including sFGFR3_Del4-C253S orsFGFR3_Del4-D3 was then loaded on an equilibrated column at 20 mL/min,after adjusting the sample's conductivity to 14 mS/cm (ÄKTA™ pure 25 (GEHealthcare)). Columns used were HiPrep Q FF 26/10 (GE Healthcare) with abed volume of 53 mL. The binding buffer was 1×PBS and the elution bufferwas PBS 1×+1 M NaCl. The column was washed with four column volumes of1×PBS. Elution of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 was performed bytwo steps of 5% NaCl and 10% NaCl using four column volumes of each.Both 5% NaCl and 10% NaCl were pooled and concentrated by cross flowfiltration (ÄKTA™ flux, GE Healthcare). The remaining volume was thenconcentrated on a 30 kDa filter by centrifugation at 4° C., 3,900 g for10 min (MILLLIPORE® UFC903024 AMICON® Ultra-15 Centrifugal FilterConcentrator). For size exclusion chromatography, the remaining volumewas loaded on a HiLoad 26/600 SUPERDEX™ 200 prep grade (28-9893-36, GEHealthcare) with a bed volume of 320 mL. Loading volume did not exceed12.8 mL. Elution was performed in 1×PBS.

Example 3: Kinetic Assays and Dissociation Constant (K_(d)) Measurementsof sFGFR3 Polypeptides

Calibration Free Concentration Analysis and kinetic assays ofsFGFR3_Del4-C253S and sFGFR3_Del4-D3 were performed with a Sensor ChipCM5 (GE Healthcare). Human FGF2 (hFGF2) was covalently immobilized tothe Sensor Chip CM5 at a level of about 5000 RU by amine coupling. Toachieve 5000 RU, hFGF2 was immobilized for 420 seconds at a flow rate 10μI/min and a concentration 25 μg/ml. Running buffer was HBS-EP+ Buffer(GE Healthcare). Regeneration buffer was 100 mM sodium acetate with 2Msodium chloride pH 4.5. FGF binding, dissociation constant (K_(d))measurements, and kinetic parameters were determined by Surface PlasmonResonance using a BIACORE™ T200 (GE Healthcare). The model used forkinetic assays and K_(d) determination was a 1:1 binding algorithm.

Example 4: Proliferation Assays of sFGFR3 Polypeptides

Both ATDC5 and ATDC5 FGFR3^(G380R) cell lines were seeded at a densityof 25,000 cells/cm² in NUNC™ MICROWELL™ 96-Well Optical-Bottom Plateswith Polymer Base (ThermoFisher Scientific, Catalog No. 165305). After a24 hour incubation period, cells were depleted for 48 hour in 0.5% BSAand then stimulated for 72 hour with sFGFR3_Del4-C253S or sFGFR3_Del4-D3with and without hFGF2 (Peprotech). Cell proliferation was then measuredusing the CyQUANT® Direct Cell Proliferation Assay (Molecular Probes,Catalog No. C35012). After stimulation, 10 μL of CyQUANT® Direct CellProliferation (Invitrogen; 1 mL 1×PBS, 250 μL background suppressor, and50 μL nuclear stain) was added per well. ATDC5 and ATDC5 FGFR3^(G380R)cells were then incubated at room temperature in the dark for 2 hours.

Fluorescence was read using the VARIOSKAN™ LUX multimode microplatereader (ThermoFisher Scientific).

Example 5: Luciferase Assays of sFGFR3 Polypeptides

Serum Response Element-Luciferase (SRE-Luc) HEK cells expressingFGFR3^(G380R) were seeded at a density of 100,000 cells/cm² in astandard culture 96 well plate. Cells were then depleted for 24 hourswith 0.5% heat inactivated Fetal Bovine Serum (hiFBS), before beingtreated with sFGFR3_Del4-D3 at concentrations of 0 nm, 70 nm, and 280 nmwith or without 1 ng/ml of hFGF2 for 24 h. The culture plate wasequilibrated to room temperature for 15 minutes prior to adding 100 μLper well of Firefly Luc One-Step Glow Assay Working Solution(ThermoFisher Scientific, Catalog No. 16197), then shaken at 600 rpm for3 minutes. The plate was incubated at room temperature for 10 minutesand each cell lysate was transferred to a white opaque 96 well plate toincrease luminescence signal and decrease cross contamination. Theluminescence signal was read using the VARIOSKAN™ LUX multimodemicroplate reader (Thermo Fisher Scientific).

Example 6: In Vivo Efficacy Study of sFGFR3 Polypeptides

Experiments were performed on transgenic Fgfr3^(ach/+) animals in whichexpression of the mutant FGFR3 is driven by the Col2a1promoter/enhancer. Mice were exposed to a 12 hour light/dark cycle andhad free access to standard laboratory food and water. Genotypes wereverified by PCR of genomic DNA using the primers5′-AGGTGGCCTTTGACACCTACCAGG-3′ (SEQ ID NO: 30) and5′-TCTGTTGTGTTTCCTCCCTGTTGG-3′ (SEQ ID NO: 31), which amplify 360 bp ofthe FGFR3 transgene.

sFGFR3_Del4-D3 produced using CHO cells was evaluated at a subcutaneousdose of 0.25 mg/kg twice weekly. At day 3, all newborn mice from asingle litter received the same dose. Control litters received 10 μl ofPBS (vehicle). Thereafter, subcutaneous injections of sFGFR3_Del4-D3(0.25 mg/kg) were administered twice a week for three weeks,alternatively on the left and right sides of the back. Mice wereobserved daily with particular attention to locomotion and urinationalterations. Breeding was performed to generate litters with half wildtype and half heterozygous Fgfr3^(ach/+) mice. To avoid bias due tophenotype penetrance variations, experiments were performed on at leasttwo litters (one treated and one control) from the same breeders.Previous data indicated there was no statistical difference betweenmales and females, and thus, males and females were considered one groupfor all analyses.

At day 22, all animals were sacrificed by lethal injection ofpentobarbital, and gender was determined. All subsequent measurementsand analyses were performed without knowledge of mice genotype to avoidinvestigator bias. Genotyping was performed at the end of the study toreveal the correspondence of data with a specific genotype. Sinceachondroplasia is a disease with phenotypic variability, all animalswere included in the study. Animals dead before day 22 were used toinvestigate the impact of treatment on premature death. Survivinganimals at day 22 were used for all analyses. All experiments and datameasurements were performed by blinded experimenters at all time points.

Following sacrifice at day 22, body weights were measured. Cadavers werecarefully skinned, eviscerated, and skeletal measurements were performedbased on X-rays. Organs were harvested, weighed, and stored in 10%formalin for further histological analysis using standardparaffin-embedded techniques. Organs were then observed for macroscopicabnormalities, such as modification of color or texture and presence ofnodules. The Principles of Laboratory Animal Care (NIH publication no.85-23, revised 1985; grants1.nih.gov/grants/olaw/references/phspol.htm)and the European commission guidelines for the protection of animalsused for scientific purposes(ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm) werefollowed during all animal experiments. All procedures were approved bythe Institutional Ethic Committee for the use of Laboratory Animals(CIEPAL Azur) (approval #NCE-2012-52).

Example 7: The Cell Line Used to Produce sFGFR3 Polypeptides Did notImpact Activity

The FGF2 binding activity, Kd, and effect on cellular signaling ofsFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1), andsFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10) produced in suspension HEK 293 cellsor CHO cells were compared. HEK 293 cells or CHO cells differ inpost-translation modification of proteins. Expression of the sFGFR3polypeptides in different cell lines did not impact Kd, bindingactivity, or the effect of the sFGFR3 polypeptides on intracellularsignaling inhibition (FIGS. 1A-1D).

Example 8: Improved Production of sFGFR3_Del4-C2538 and sFGFR3_Del4-D3

The sFGFR3 polypeptides of sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10) were each modified toinclude either an amino acid substitution of a cysteine residue with aserine residue at position 253 or an extended Ig-like C2-type domain 3(SEQ ID NO: 33). These modifications of sFGFR3_Del1 andsFGFR3_Del4-LK1-LK2 had no or minimal effect on production of the sFGFR3polypeptides, since aggregation was still visible (FIGS. 2A and 2B,respectively). Surprisingly, modification of sFGFR3_Del4 to includeeither an amino acid substitution of a cysteine residue with a serineresidue at position 253 (sFGFR3_Del4-C253S) or an extended Ig-likeC2-type domain 3 (SEQ ID NO: 33)) improved production of the sFGFR3polypeptides. In particular, there was minimal aggregation ofsFGFR3_Del4-C253S and sFGFR3_Del4-D3 under both reducing andnon-reducing conditions (FIG. 2C). The inclusion of C253S or D3 alsoresulted in a relative increase in production compared to sFGFR3_Del4, atwo-fold increase in sFGFR3_Del4-C253S production and a 3-fold increasein sFGFR3_Del4-D3 production.

Additionally, sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3exhibited similar Kd and were not affected by cell type specific changesin post translational modifications. In Expi CHO cells, the Kd ofsFGFR3_Del4 was 0.8 nM, the Kd of sFGFR3_Del4-C253S was 0.6 nM, and theKd of sFGFR3_Del4-D3 was 0.7 nM (FIG. 3A and Table 1).

TABLE 1 Dissociation constant (Kd) of sFGFR3 polypeptides. sFGFR3Polypeptide Kd (nM) sFGFR3_Del4 0.8 sFGFR3_Del4-C253S 0.6 sFGFR3_Del4-D30.7

Example 9: sFGFR3_Del4-C253S and sFGFR3_Del4-D3 are Equally Active InVitro

sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 restoredproliferation of ATDC5 cells genetically modified to overexpress theFGFR3^(ach) mutation (ATDC5 FGFR3^(G380R) cell lines). At a dose of 36nM, sFGFR3_Del4 produced using HEK 293 cells increased proliferation to115.5%, sFGFR3_Del4 produced using CHO—S cells increased proliferationto 116%, sFGFR3_Del4-C253S produced using CHO—S cells increasedproliferation to 114.4%, and sFGFR3_Del4-D3 using CHO—S cells increasedproliferation to 120.1% (FIG. 3B).

sFGFR3_Del4-D3 was also tested in the FGFR3^(G380R) expressing SRE(-Luc)HEK cell line at doses of 0 nM, 70 nM, and 280 nM with or without 1ng/ml of hFGF2 (FIG. 4; n=8). Data shown in FIG. 4 are themean+/−standard error of the mean (SEM). These data followed a normallaw and have equal variance based on the D'Agostino-Pearson omnibusnormality test. Statistical comparisons with and without sFGFR3_Del4-D3were performed using a student t-test. As shown in FIG. 4,sFGFR3_Del4-D3 decreases luciferase signaling in the SRE cell line.

Example 10: sFGFR3_Del4-D3 Restores Bone Growth, Prevents Mortality, andRestores Foramen Magnum Shape in Mice with Achondroplasia

An in vivo efficacy study was performed as in Example 6 using a low dose(0.25 mg/kg) of sFGFR3_Del4-D3. A total of 60 mice were included in thevehicle group, with 32 wild type (wt) mice and 28 Fgfr3^(ach/+) mice.The treated group included 40 mice, with 19 wt mice and 21 Fgfr3^(ach/+)mice. Surprisingly, the low dose of sFGFR3_Del4-D3 almost completelyprevented the premature death of mice with achondroplasia (FIG. 5). Inthe control group, 53.6% of the Fgfr3^(ach/+) mice died before weaning,whereas only 4.8% of mice in the treated group died before day 22 and20% of mice died following treatment with sFGFR3_Del1 at 0.25 mg/kg(Table 2; see also Garcia et al. Sci. Transl. Med. 5:203ra124, 2013,hereby incorporated by reference in its entirety).

sFGFR3_Del4-D3 also partially restored bone growth with correction ofthe initial discrepancy between wt and Fgfr3^(ach/+) mice on the axialand appendicular skeleton (Table 2). In contrast to prior results oftreatment with a low dose of sFGFR3 Del1, treatment with low dose ofsFGFR3_Del4-D3 restored normal foramen magnum shape.

TABLE 2 In vivo results of administering a high dose of sFGFR3_Del1, alow dose of sFGFR3_Del1, and a low dose of sFGFR3_Del4-D3 to mice withachondroplasia 2.5 mg/kg 0.25 mg/kg sFGFR3_Del1 sFGFR3_Del1 0.25 mg/kg(Garcia et al.) (Garcia et al.) sFGFR3_Del4-D3 Mortality    12%   20% 4.8% Axial correction    77%   24%   10% Appendicular 150-215% 18-42%11-42% correction Foramen shape Not determined Not determined   111%correction (ratio W/H)

Example 11: Treatment of Achondroplasia by Administration ofsFGFR3_Del4-C253S

A human patient (e.g., an infant, child, adolescent, or adult) sufferingfrom achondroplasia can be treated by administering sFGFR3_Del4-C253S(FIG. 6; SEQ ID NO: 2) by an appropriate route (e.g., by subcutaneousinjection) at a particular dosage (e.g., between 0.0002 mg/kg/day toabout 20 mg/kg/day, such as 0.001 mg/kg/day to 7 mg/kg/day) over acourse of days, weeks, months, or years. The progression ofachondroplasia that is treated with sFGFR3_Del4-C253S can be monitoredby one or more of several established methods. A physician can monitorthe patient by direct observation in order to evaluate how the symptomsof achondroplasia exhibited by the patient have changed in response totreatment. For instance, a physician may monitor changes in body weight,skull length, and/or skull width of the patient over a period of time,e.g., 1, 2, 3, 4 or more times per month or per year or approximatelyevery 1, 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks over the course oftreatment with sFGFR3_Del4-C253S. Body weight and/or skull size of thepatient or changes thereof can also be determined at treatment specificevents, e.g. before and/or after administration of sFGFR3_Del4-C253S.For example, body weight and/or skull size are measured in response toadministration of sFGFR3_Del4-C253S.

Example 12: Treatment of Achondroplasia by Administration ofsFGFR3_Del4-D3

Additionally, a human patient (e.g., an infant, child, adolescent, oradult) suffering from achondroplasia can be treated by administering thesFGFR3 polypeptide of sFGFR3_Del4-D3 (SEQ ID NO: 33) by an appropriateroute (e.g., by subcutaneous injection) at a particular dosage (e.g.,between 0.0002 mg/kg/day to about 20 mg/kg/day, such as 0.001 mg/kg/dayto 7 mg/kg/day) over a course of days, weeks, months, or years. Theprogression of achondroplasia that is treated with sFGFR3_Del4-D3 can bemonitored by one or more of several established methods. A physician canmonitor the patient by direct observation in order to evaluate how thesymptoms of achondroplasia exhibited by the patient have changed inresponse to treatment. For instance, a physician may monitor changes inbody weight, skull length, and/or skull width of the patient over aperiod of time, e.g., 1, 2, 3, 4 or more times per month or per year orapproximately every 1, 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks over thecourse of treatment with sFGFR3_Del4-D3. Body weight and/or skull sizeof the patient or changes thereof can also be determined at treatmentspecific events, e.g. before and/or after administration ofsFGFR3_Del4-D3. For example, body weight and/or skull size are measuredin response to administration of sFGFR3_Del4-D3.

Example 13: Production of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S

The sFGFR3_Del4-D3 and sFGFR3_Del4-C253S polypeptides were purified asdescribed in Example 2. Modification of sFGFR3 Del4 to include either anextended Ig-like C2-type domain 3 (FGFR3_Del4-D3) or an amino acidsubstitution of a cysteine residue with a serine residue at position 253(sFGFR3_Del4-C253S) improved production of the sFGFR3 polypeptides. Inparticular, there was less than about 2% aggregation of sFGFR3_Del4-D3and sFGFR3_Del4-C253S (as observed upon loading using a concentration of2.3 mg/ml or 23 mg/ml for FGFR3_Del4-D3 and 1.5 mg/ml and 15 mg/ml ofsFGFR3_Del4-C253S) under both reducing and non-reducing conditions usingsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE;FIGS. 7A and 7B, respectively). Following production of sFGFR3_Del4-D3and sFGFR3_Del4-C253S in fed-batch cultures, the top five clones wereseparated using capillary electrophoresis to yield 0.93 to 1.0 g/L and0.98 to 1.1 g/L of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S, respectively.Viral filtration using ion-exchange chromatography resulted in a yieldof greater than 60% for both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S.

Example 14: Pharmacokinetics and Tissue Distribution of sFGFR3_Del4-D3In Vivo

In vivo studies were performed to investigate the pharmacokineticparameters of sFGFR3_Del4-D3, the uptake of sFGFR3_Del4-D3 across theblood brain barrier, and the tissue distribution of sFGFR3_Del4-D3 inkidney, liver, spleen, lung, and heart. The studies described hereinincluded four arms with five groups of C57BL/6J mice per arm and a totalof four mice (n=4) per group (Table 3). Mice were male and weighed 25 to30 grams.

TABLE 3 Overview of mice used in studies of sFGFR3_Del4-D3. sFGFR3_Del4-Tissue Arm D3 (mg/kg) Route PK BBB distribution 1 0.25 SC yes no no 22.5 SC yes no yes 3 2.5 IV yes Yes yes 4 10 SC yes no no

Group 1 was sampled at 1 minute, 15 minutes, and 30 minutes; group 2 wassampled at 4 hours; group 3 was sampled at 24 hours; group 4 was sampledat 36 hours; and group 5 was sampled at 48 hours. For Group 1, anindwelling intra-arterial catheter (PE-10) was inserted into one commoncarotid artery under isoflurane anesthesia and used for repeated bloodsampling at the 30 minute final sampling time point. For intravenousinjection, ¹²⁵I-sFGFR3_Del4-D3 was injected intravenously into thejugular vein, which was exposed by skin incision under isofluraneanesthesia. Group 1 mice remained anesthetized throughout theexperiments. Repeated blood samples (2×-50 μL) were drawn from thearterial catheter at 1 minute and 15 minutes after intravenousinjection. For groups 2 to 5, after injection of ¹²⁵I-sFGFR3_Del4-D3,the skin was closed with a surgical clip, and the mice were allowed towake up and returned to the cage. At 5 minutes before termination timefor group 3, mice were re-anesthetized and received an intravenous bolusof ³H-albumin into the jugular vein. The ³H tracer dose was targeted toyield a ratio of ¹²⁵I to ³H in blood, which is suitable for doubleisotope labeling with a lower dose at later sampling times. At theterminal sampling time (2 hours, 3 hours, 24 hours, 36 hours, and 48hours), a blood sample was collected, and the animal was euthanized. Thebrain was sampled for homogenization and determination of tissueconcentration of tracers. Endpoints of the studies includedpharmacokinetic parameters for sFGFR3_Del4-D3 (terminal half life),uptake of sFGFR3_Del4-D3 across the blood brain barrier, and the tissuedistribution of sFGFR3_Del4-D3 in kidney, liver, spleen, lung, andheart.

Example 15: Thermal and Plasma Stability of sFGFR3_Del4-D3 andsFGFR3_Del4-C253S

The thermal stability of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S in mouseplasma was investigated using differential scanning colorimetry. ForsFGFR3_Del4-D3, two buffers (20 mM phosphate, 40 mM NaCl, pH 7.5, and 20mM citrate, 40 mM NaCl, pH 6.5) were added to polypeptide samples. ForsFGFR3_Del4-C253S, two buffers (20 mM phosphate, 40 mM NaCl, pH 7.5, and40 mM citrate, 40 mM NaCl, pH 6.5) were added to polypeptide samples.The melting temperature (T_(m)) for sFGFR3_Del4-C253S in the 20 mMphosphate, 40 mM NaCl, pH 7.5 buffer was 52° C. and 56° C., and theT_(m) for sFGFR3_Del4-C253S in the 40 mM citrate, 40 mM NaCl, pH 6.5buffer was 55° C. and 60° C. (FIG. 8A). For sFGFR3_Del4-D3, two buffers(20 mM phosphate, 40 mM NaCl, pH 7.5, and 20 mM citrate, 40 mM NaCl, pH6.5) were added to polypeptide samples. The T_(m) for sFGFR3_Del4-D3 inthe 20 mM phosphate, 40 mM NaCl, pH 7.5 buffer was 50° C. and 54° C.,and the T_(m) for sFGFR3_Del4-D3 in the 20 mM citrate, 40 mM NaCl, pH6.5 buffer was 53° C. and 58° C. (FIG. 8B). These results indicate thatboth sFGFR3_Del4-D3 and sFGFR3_Del4-C253S show two domains ofpolypeptide stability and unfolding.

The ex vivo plasma stability of sFGFR3_Del4-D3 with a Histidine tag wasdetermined by labeling purified sFGFR3_Del4-D3 with ¹²⁵I-tracer usingthe Bolton-Hunter method, followed by purification on PD-10 (Sephadex®G-25) columns. The trichloroacetic acid (TCA) precipitability of peakfractions was also determined to confirm stability of the ¹²⁵I-tracer.Mouse plasma (n=4) pre-warmed to 37° C. was spiked with the¹²⁵I-sFGFR3_Del4-D3 to a concentration of ˜10 cpm/mL and then vortexed.The plasma samples were incubated with the ¹²⁵I-sFGFR3_Del4-D3 in anEppendorf ThermoMixer® under gentle rotation (300 rpm). Aliquots werethen collected for TCA precipitation (10 μl sample and 100 μl 2% BSA)and for injection onto an Fast Performance Liquid Chromatography (FPLC)column (20 μl sample and 150 μl 10 mM PBS, pH 7.4) at intervals of 0,30, 60, 120, 180, and 360 minutes. Aliquots were stored on ice until TCAprecipitation or FPLC injection was performed.

For TCA precipitation, 1 mL ice cold 10% TCA was added to plasmasamples, incubated for 10 minutes on ice, centrifuged at 4,000 g for 5minutes, and then the supernatant and pellet were separated and bothwere counted in a gamma counter. For evaluation of the ex vivo plasmastability, 100 μl of the sample was injected on an FPLC column(Superdex® 200 10/300 GL) and eluted at a rate of 0.75 ml/min for 1.5column volumes. Fractions of 1 ml were collected from the column andthen measured in a gamma counter. The plasma stability of sFGFR3_Del4-D3at 37° C. was determined to be 95% at 0 minutes, 95% at 2 hours, and˜92% at 24 hours with only minor aggregation (FIG. 9A).

The in vivo stability of sFGFR3_Del4-D3 in plasma after administrationby intravenous and subcutaneous injection was also determined.sFGFR3_Del4-D3 was labeled with ¹²⁵I-tracer using the Bolton-Huntermethod, followed by purification on PD-10 (Sephadex® G-25) columns. The¹²⁵I-labeled sFGFR3_Del4-D3 (10 μCi in ˜50 μL PBS) was administered byintravenous or subcutaneous injection into anesthetized C57BI/6 mice.The ¹²⁵I-tracer protein dose (approximately 0.1 mg/kg) was complementedwith unlabeled protein to a total dose of 2.5 mg/kg. Rat serum albuminused as a vascular marker was labeled with [³H]-NSP(N-succininidyl[2,3-³H]Propionate; Perkin Elmer) and purified on PD-10(Sephadex® G25) columns.

For the stability of sFGFR3_Del4-D3 in plasma after intravenous bolusinjection, FPLC elution profiles showed no degradation products inplasma up to 15 minutes (FIGS. 9B-9D). At 30 minutes afteradministration of sFGFR3_Del4-D3, a small amount of low molecular weightdegradation products appeared, which increased by 2 hours, but largelydisappeared by 24 hours. For the stability of sFGFR3_Del4-D3 in plasmaafter subcutaneous injection, FPLC elution profiles showed somedegradation products in plasma at 30 minutes, with increased degradationby 2 hours and 4 hours (FIGS. 9E-9G). The low amount of tracer left inplasma after 24 hours appears largely as the intact sFGFR3_Del4-D3polypeptide. Chromatograms in FIGS. 9C-9D and 9F-9G are presented asnormalized to the highest peak in each individual run for easiercomparison of the elution patterns.

Example 16: Ligand Binding Activity of sFGFR3_Del4-D3 andsFGFR3_Del4-C253S

Experiments were performed to characterize the binding affinity ofsFGFR3_Del4-D3 and sFGFR3_Del4-C253S for human FGF2. The dissociationconstant (Kd) of sFGFR3_Del4-D3 and Kd of sFGFR3_Del4-C253S for FGF2were determined as described in Example 3 with a regeneration buffer of20 mM phosphate, 40 mM NaCl, pH 7.5. Concentrations of 13 nM, 6.5 nM,3.25 nM, and 1.75 nM were tested for both sFGFR3_Del4-D3 andsFGFR3_Del4-C253S. The Kd of sFGFR3_Del4-D3 was determined to be ˜3.6nm, and the Kd of sFGFR3_Del4-C253S was determined to be ˜6.9 nm. Theseresults indicate that sFGFR3_Del4-D3 and sFGFR3_Del4-C253S have bindingactivity for FGF2 in the low nM range.

Example 17: sFGFR3_Del4-D3 and sFGFR3_Del4-C253S Exhibit FunctionalActivity In Vitro

Functional activity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S was testedusing a proliferation assay. Proliferation assays using ATDC5 cellsgenetically modified to overexpress the FGFR3^(ach) mutation (ATDC5FGFR3^(G380R) cell lines) were performed as described in Example 4 withconcentrations of 1 ug/ml, 10 ug/ml, and 50 ug/ml for sFGFR3_Del4-D3 andsFGFR3_Del4-C253S. At each of these concentrations, sFGFR3_Del4-C253Sand sFGFR3_Del4-D3 restored proliferation of the FGFR3^(G380R) cells(FIGS. 10A and 10B). The EC50 was determined to be about 10 nM for bothsFGFR3_Del4-D3 and sFGFR3_Del4-C253S based on a concentration of 1ug/ml. These results indicate that sFGFR3_Del4-D3 and sFGFR3_Del4-C253Sare biologically active in the low nM range.

Example 18: Pharmacokinetic Profile of sFGFR3_Del4-D3 andsFGFR3_Del4-C253S

The pharmacokinetic (PK) profile of sFGFR3_Del4-D3 administeredsubcutaneously or intravenously at a dose of 2.5 mg/kg was used todetermine the terminal elimination half-life of sFGFR3_Del4-D3 (FIG.11). Samples were collected at 30 minutes, 2 hours, 4 hours, 8 hours, 24hours, 36 hours, and 48 hours for mice administered sFGFR3_Del4-D3subcutaneously. Samples were collected at 1 minute, 15 minutes, 30minutes, 2 hours, 24 hours, and 36 hours for mice administeredsFGFR3_Del4-D3 intravenously. The subcutaneous terminal eliminationhalf-life of 2.5 mg/kg sFGFR3_Del4-D3 was ˜20 hours, while theintravenous terminal elimination half-life of 2.5 mg/kg sFGFR3_Del4-D3was ˜7 hours. From the PK profile, the T_(max) was ˜8 hours, the C_(max)was ˜4.5 nM, and the estimated bioavailability was ˜30% for 2.5 mg/kgsFGFR3_Del4-D3 administered subcutaneously. There was rapid clearance ofsFGFR3_Del4-D3 administered intravenously during the a phase followed bya slower β phase clearance, with a similar intravenous PK profile forsFGFR3_Del4-C253S.

Example 19: The Kidney and Liver are the Main Clearance Routes ofsFGFR3_Del4-D3

Clearance of sFGFR3_Del4-D3 was evaluated in kidney, liver, spleen,lung, and heart tissue after 30 minutes, 120 minutes, and 1440 minutesfollowing intravenous administration of 2.5 mg/kg sFGFR3_Del4-D3 andafter 30 minutes, 120 minutes, 240 minutes, 480 minutes, and 1440minutes following subcutaneous administration of 2.5 mg/kgsFGFR3_Del4-D3. The liver and kidney were the major route ofsFGFR3_Del4-D3 clearance for intravenous administration (FIG. 12). Thekidney was the major route of sFGFR3_Del4-D3 clearance for subcutaneousadministration (FIG. 13).

Example 20: sFGFR3_Del4-D3 does not Cross the Blood Brain Barrier

Pharmacokinetic studies were also performed to determine the uptake ofsFGFR3_Del4-D3 across the blood brain barrier in wild-type mice. Afterintravenous bolus injection, brain tissue uptake of sFGFR3_Del4-D3 wasmeasured at three time points (30 minutes, 2 hours, and 24 hours).sFGFR3_Del4-D3 was injected as radiolabeled tracer (¹²⁵I-sFGFR3_Del4-D3)with 2.5 mg/kg unlabeled sFGFR3_Del4-D3. The injected dose of¹²⁵I-sFGFR3_Del4-D3 was about 10 μCi per animal, which corresponds toless than 0.1 mg/kg. After euthanizing the mice at 30 minutes, 2 hours,and 24 hours, the concentration of ¹²⁵I-sFGFR3_Del4-D3 in organs andplasma was measured by liquid scintillation counting.

The ¹²⁵I-sFGFR3_Del4-D3 concentration was corrected for metabolism inplasma and in brain samples by measuring the fraction of trichloroaceticacid (TCA) precipitable material (e.g., intact tracer). The validity ofthe TCA correction was also confirmed by injecting samples on a sizeexclusion fast protein liquid chromatography (FPLC) column. The organconcentration of ¹²⁵I-sFGFR3_Del4-D3 was corrected for intravascularcontent (V₀) by injecting radiolabeled albumin (³H-RSA) shortly beforesacrificing the animal. The apparent organ volume of distribution of RSArepresents V₀. The dose of albumin was negligible (on the order of 1% ofthe physiological concentration). For all organs other than the brain,the concentrations were calculated by subtracting the vascular contentand taking into account the TCA precipitable fraction in plasma.However, no correction was made for the uptake of degraded material intothese organs other than the brain because no TCA precipitation wasperformed.

The brain concentrations were calculated by the following formula:C_(brain(corr.))=[V_(d)(sFGFR3_Del4-D3)−V₀]×C_(plasma (terminal)), inwhich V_(d)(sFGFR3_Del4-D3) is the volume of distribution ofsFGFR3_Del4-D3 in brain (calculated as C_(brain)/C_(plasma)), V₀ is thevolume of albumin distributed in the brain, and C_(plasma(terminal)) isthe plasma concentration of sFGFR3_Del4-D3 at the terminal samplingtime. All concentrations were expressed as the percent of injected doseper gram or ml (% ID/g or % ID/mL), respectively, and the dose of theintravenous bolus equals 100%. These values can be converted to [mg/g]or [mg/mL] by multiplication with the injected dose: (body weight ing/1000 g)×2.5 mg. All body weights were in the range of 25 g-30 g.

There was no detectable brain uptake of ¹²⁵I-sFGFR3_Del4-D3, asindicated by corrected brain concentrations (after correction forvascular content and degradation (TCA precipitability)) at at any of themeasured time points (FIG. 14A). Additionally, the V_(d) of RSA (=V0)and ¹²⁵I-sFGFR3_Del4-D3 was not significantly different at any of themeasured time points (30 minutes, 2 hours, and 24 hours) as determinedby a paired t-test (FIG. 14B). In conclusion, there is no measurableuptake of sFGFR3_Del4-D3 into brain tissue of mice at 30 minutes, 2hours, and 24 hours at a dose of 2.5 mg/kg injected as an intravenousbolus.

Example 21: In Vivo Efficacy of sFGFR3_Del4-D3 for the Treatment ofAchondroplasia

sFGFR3_Del4-D3 and sFGFR3_Del4-C253S were each evaluated at asubcutaneous dose of 2.5 mg/kg once or twice weekly or 10 mg/kg twiceweekly. Breeding was performed to generate 30 litters with half wildtype and half heterozygous Fgfr3^(ach/+) mice (Table 4).

TABLE 4 Subcutaneous administration of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S to wild type (WT) and Fgfr3^(ach/+) mice. PBS 2.5 mg 2.5 mg 10 mg(pooled) 1X week 2X week 2X week sFGFR3_Del4-D3 WT 65 26 22 23Fgfr3^(ach/+) 43 26 25 30 total N = 260 sFGFR3_Del4- C253S WT 65 26 2223 Fgfr3^(ach/+) 27 22 18 28 total N = 231 % survival 62.8 84.6 72.093.3 % mortality 37.2 15.4 28.0  6.7

At day 3, all newborn mice from a single litter received the same dose.Control litters received 10 μl of PBS (vehicle). Thereafter,subcutaneous injections of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S wereadministered at doses of 2.5 mg/kg once or twice weekly or 10 mg/kgtwice a week for three weeks, alternatively on the left and right sidesof the back. Mice were observed daily with particular attention tolocomotion and urination alterations and weighed on days of injection.Mice with complications were observed twice a day for surveillance.Previous data indicated there was no statistical difference betweenmales and females, and thus, males and females were considered one groupfor all analyses.

At day 22, all animals were sacrificed by lethal injection ofpentobarbital, and gender was determined. All subsequent measurementsand analyses were performed without knowledge of mice genotype to avoidinvestigator bias. Genotyping was performed at the end of the study toreveal the correspondence of data with a specific genotype. Sinceachondroplasia is a disease with phenotypic variability, all animalswere included in the study. Animals dead before day 22 were used toinvestigate the impact of treatment on premature death. Survivinganimals at day 22 were used for all analyses. All experiments and datameasurements were performed by blinded experimenters at all time points.

Subcutaneous administration of sFGFR3_Del4-D3 at 2.5 mg/kg once or twiceweekly or 10 mg/kg twice weekly increased survival of Fgfr3^(ach/+) micerelative to Fgfr3^(ach/+) mice receiving PBS (FIG. 15 and Table 4). Inparticular, administration of 10 mg/kg sFGFR3_Del4-D3 twice weeklyresulted in 93% survival of Fgfr3^(ach/+) mice, administration of 2.5mg/kg sFGFR3_Del4-D3 once weekly resulted in 84% survival inFgfr3^(ach/+) mice, and administration of 2.5 mg/kg sFGFR3_Del4-D3 twiceweekly resulted in 72% survival in Fgfr3^(ach/+) mice, while thesurvival of Fgfr3^(ach/+) mice receiving PBS was 62.8%. The mortality ofFgfr3^(ach/+) mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly was6.7%, the mortality of Fgfr3^(ach/+) mice administered 2.5 mg/kgsFGFR3_Del4-D3 once weekly was 15.4%, the mortality of Fgfr3^(ach/+)mice administered 2.5 mg/kg sFGFR3_Del4-D3 twice weekly was 28.0%, andthe mortality of Fgfr3^(ach/+) mice administered PBS was 37.2%.Statistical analysis of Fgfr3^(ach/+) mice survival following treatmentwith sFGFR3_Del4-D3 was performed using the Agostino and Pearson omnibusnormality test following by a t-test. All investigated groups passed thenormality tests. The P-values from these analyses are shown below, inwhich * represent a P-value of <0.05 and *** represents a P-value of<0.001 (Table 5).

TABLE 5 P-values for subcutaneous administration of sFGFR3_Del4-D3 towild type (WT) and Fgfr3^(ach/+) mice. Group Comparison P Value Wt vsach *** Fgfr3^(ach/+) PBS vs Fgfr3^(ach/+) 2.5 mg/kg, 1x ***Fgfr3^(ach/+) PBS vs Fgfr3^(ach/+) 2.5 mg/kg, 2x * Fgfr3^(ach/+) PBS vsFgfr3^(ach/+) 10 mg/kg, 2x *** Wt PBS vs Fgfr3^(ach/+) 10 mg/kg, 2x ns

Subcutaneous administration of sFGFR3_Del4-D3 at 2.5 mg/kg once or twiceweekly or 10 mg/kg twice weekly also decreased the severity andfrequency of locomotor problems and complications in abdominal breathingin Fgfr3^(ach/+) mice relative to Fgfr3^(ach/+) mice receiving PBS (FIG.16). In particular, locomotor problems decreased the most inFgfr3^(ach/+) mice administered subcutaneously 10 mg/kg sFGFR3_Del4-D3twice weekly followed by mice administered sFGFR3_Del4-D3 2.5 mg/kgtwice weekly and mice administered sFGFR3_Del4-D3 2.5 mg/kg once weekly.Complications in abdominal breathing decreased the most in Fgfr3^(ach/+)mice administered subcutaneously 10 mg/kg sFGFR3_Del4-D3 twice weeklyfollowed by mice administered sFGFR3_Del4-D3 2.5 mg/kg once weekly andthen mice administered sFGFR3_Del4-D3 2.5 mg/kg twice weekly. Theseresults show that sFGFR3_Del4-D3 reduces symptoms of achondroplasia inFgfr3^(ach/+) mice.

Subcutaneous administration of sFGFR3_Del4-D3 also significantlyincreased total body length, including axial length and tail length, andlong bones (p=0.07) in Fgfr3^(ach/+) mice receiving 2.5 mg/kgsFGFR3_Del4-D3 once or twice weekly or 10 mg/kg sFGFR3_Del4-D3 twiceweekly relative to Fgfr3^(ach/+) mice receiving PBS (FIGS. 17A-17C).Tail and body length (axial length) were measured using the same digitalcaliper on whole skeletons. Tibia length was measured on digital X-rays.Administration of 10 mg/kg sFGFR3_Del4-D3 twice weekly resulted in 51%axial correction (body and tail length) of Fgfr3^(ach/+) mice, followedby 43% axial correction in Fgfr3^(ach/+) receiving 2.5 mg/kgsFGFR3_Del4-D3 twice weekly, and 39% axial correction in Fgfr3^(ach/+)mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once weekly. Increases in boneand body length were also evident from x-ray radiographs ofFgfr3^(ach/+) mice administered 2.5 mg/kg or 10 mg/kg sFGFR3_Del4-D3twice weekly relative to Fgfr3^(ach/+) mice receiving PBS (FIG. 17D).Administration of 10 mg/kg sFGFR3_Del4-D3 twice weekly resulted in 86%appendicular correction (tibia and femur length) of Fgfr3^(ach/+) mice,followed by 68% appendicular correction in Fgfr3^(ach/+) receiving 2.5mg/kg sFGFR3_Del4-D3 twice weekly and 54% appendicular correction inFgfr3^(ach/+) mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once weekly.

Subcutaneous administration of sFGFR3_Del4-D3 also resulted in adose-dependent improvement in cranial ratio (length/width (L/VV)) inFgfr3^(ach/+) mice relative to Fgfr3^(ach/+) mice receiving PBS (FIG.18A). Fgfr3^(ach/+) mice subcutaneously administered 10 mg/kgsFGFR3_Del4-D3 twice weekly exhibited the greatest improvement in thecranium ratio (LAN), followed by Fgfr3^(ach/+) mice administered 2 mg/kgsFGFR3_Del4-D3 twice weekly and Fgfr3^(ach/+) mice administered 2 mg/kgsFGFR3_Del4-D3 once weekly. In particular, administration of 10 mg/kgsFGFR3_Del4-D3 twice weekly resulted in 37% skull shape correction (L/VVratio) of Fgfr3^(ach/+) mice, followed by 29% skull shape correction inFgfr3^(ach/+) receiving 2.5 mg/kg sFGFR3_Del4-D3 twice weekly and 19%skull shape correction in Fgfr3^(ach/+) mice receiving 2.5 mg/kgsFGFR3_Del4-D3 once weekly. Improvements in the cranial ratio were alsoevident from x-ray radiographs of Fgfr3^(ach/+) mice administered 10mg/kg sFGFR3_Del4-D3 relative to Fgfr3^(ach/+) mice receiving PBS (FIG.18B). Bone measurements (presented in mm and mean±SEM) for body length,tail, femur, tibia, and cranial ratio are shown below (Table 6). Theseresults indicate the dose-dependent in vivo efficacy of sFGFR3_Del4-D3as demonstrated by increased survival, reduced number of complications,increased bone growth, and improvements in skeletal proportions ofFgfr3^(ach/+) mice.

TABLE 6 Bone measurements (presented in mm and mean ± SEM) for bodylength, tail, femur, tibia, and cranial ratio of WT and Fgfr3^(ach/+)mice administered subcutaneously sFGFR3_Del4-D3. Efficacy ofsFGFR3_Del4-D3 PBS in Fgfr3^(ach/+) 2.5 mg/kg 2.5 mg/kg 10 mg/kg WT miceonce weekly twice weekly twice weekly Body length 144.8 ± 0.53 129.2 ±1.98   135 ± 1.48 135.5 ± 1.75 135.2 ± 1.58 Tail 77.65 ± 0.39 70.25 ±1.1  73.37 ± 1.66 73.69 ± 1.5  74.95 ± 0.91 Femur 10.94 ± 0.05 10.14 ±0.13 10.47 ± 0.08 10.58 ± 0.09 10.63 ± 0.10 Tibia 14.19 ± 0.05 13.67 ±0.14 14.02 ± 0.10 14.09 ± 0.12 14.25 ± 0.12 Cranial ratio  1.99 ± 0.01 1.79 ± 0.01  1.83 ± 0.02  1.85 ± 0.01  1.86 ± 0.02

Additionally, comparison of the bone measurements for Fgfr3^(ach/+) miceadministered sFGFR3 Del1 at a dosage of 2.5 mg/kg twice weekly show thatadministration sFGFR3_Del4-D3 at a dosage of 2.5 mg/kg twice weekly wascomparable to or more effective in increasing the bone, tail, femur, andtibia length and improving the cranial ratio of Fgfr3^(ach/+) mice(Table 7). In particular, the body length of Fgfr3^(ach/+) miceadministered sFGFR3_Del4-D3 improved to 135.5±1.75 mm relative to134.4±1.17 mm for Fgfr3^(ach/+) mice administered sFGFR3_Del1; the taillength of Fgfr3^(ach/+) mice administered sFGFR3_Del4-D3 improved to73.69±1.5 mm relative to 71.58±0.86 mm for Fgfr3^(ach/+) miceadministered sFGFR3_Del1; the femur length of Fgfr3^(ach/+) miceadministered sFGFR3_Del4-D3 improved to 10.58±0.09 mm relative to10.01±0.06 mm for Fgfr3^(ach/+) mice administered sFGFR3_Del1; the tibialength of Fgfr3^(ach/+) mice administered sFGFR3_Del4-D3 improved to14.09±0.12 mm relative to 13.27±0.31 mm for Fgfr3^(ach/+) miceadministered sFGFR3_Del1; and the cranial ratio of Fgfr3^(ach/+) miceadministered sFGFR3_Del4-D3 improved to 1.85±0.01 mm relative to1.81±0.02 mm for Fgfr3^(ach/+) mice administered sFGFR3 Del1.

TABLE 7 Bone measurements (presented in mm and mean ± SEM) for bodylength, tail, femur, tibia, and cranial ratio of WT and Fgfr3^(ach/+)mice administered subcutaneously sFGFR3_Del1 (data described in Garciaet al. Sci. Transl. Med. 5: 203ra124, 2013). Efficacy of sFGFR3_Del1 PBSin Fgfr3^(ach/+) 0.25 mg/kg 2.5 mg/kg WT mice twice weekly twice weeklybody length 133.9 ± 0.8  118.5 ± 1.76 132.4 ± 1.26 134.4 ± 1.17 tail 71.9 ± 0.49 64.48 ± 1.1  71.05 ± 0.99 71.58 ± 0.86 femur 10.05 ± 0.17 9.67 ± 0.16  9.85 ± 0.10 10.01 ± 0.06 tibia 13.43 ± 0.19 12.62 ± 0.1812.87 ± 0.14 13.27 ± 0.31 cranial ratio  1.94 ± 0.01  1.75 ± 0.01  1.77± 0.02  1.81 ± 0.02

Example 22: No Organ Toxicity Associated with Administration ofsFGFR3_Del4-D3

Histopathological studies were performed to characterize organ toxicityassociated with sFGFR3_Del4-D3 administration. Wild type mice (6 malesand 6 females per dose) were administered PBS, 2.5 mg/kg sFGFR3_Del4-D3once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, or 10 mg/kgsFGFR3_Del4-D3 twice weekly. Organs investigated included the kidney,skin, salivary glands, mandibular lymph nodes, gall bladder, spleen,pancreas, lungs, heart, aorta, jejunum, colon, and liver. There were nohistopathological results indicating organ toxicity in wild-type miceadministered any of the doses of sFGFR3_Del4-D3. These results indicatethat there was no toxicity associated with administration ofsFGFR3_Del4-D3 up 10 mg/kg twice weekly.

Example 23: Determination of Binding Affinity of sFGFR3_Del4-D3 toFibroblast Growth Factors

We determined that sFGFR3_Del4-D3 binds to Fibroblast Growth Factors(FGF) ligands and acts as a decoy to prevent the binding of FGFs to themembrane bound FGFR3. Surface Plasmon Resonance was performed using aBIACORE™ T200 (GE Healthcare) to determine the K_(d) values fordifferent human FGFs (hFGFs) binding to immobilized sFGFR3_Del4-D3. Inparticular, K_(d) values for the paracrine hFGFs of hFGF1 (FIG. 19A),hFGF2 (FIG. 19B), hFGF9 (FIG. 19C), and hFGF18 (FIG. 19D) and theendocrine hFGFs of hFGF19 (FIG. 19E) and hFGF21 (FIG. 19F) weredetermined. All four paracrine FGF ligands bound sFGFR3_Del4-D3 withnanomolar (nM) affinity (Table 8).

TABLE 8 Summary of Kd determination and values for human, paracrine FGFs(hFGF1, hFGF2, hFGF9, and hFGF18) and human, endocrine FGFs (hFGF19 andhFGF21). Chi² K_(D) (M) Chi² k_(a1) k_(a2) K_(D) (M) (RU²) Steady (RU²)Binding (1/Ms) (1/Ms) k_(d1) (1/s) k_(d2) (1/s) Kinetic average stateaverage Paracrine FGFs FGF1 2:1 binding 2.0* 1.2* 1610 6.4* 2.6* 10⁻⁹0.138 5.7* 10⁻⁹ 0.247 & 10⁺¹¹ 10⁻³ 10⁻⁴ (+/−1.9* (+/−2.1* steady 10⁻⁹, n= 3) 10⁻⁹, n = 3) state FGF2 1:1 9.0* 4.75* 6.1* 10⁻¹⁰ 13.6 binding 10⁺⁵10⁻⁴ (+/−1.7*10- FGF9 2:1 2.3* 3.0* 2.6* 3.6* 1.8* 10⁻⁹ 0.14 3.6* 10⁻⁹0.25 binding 10⁺⁶ 10⁻² 10⁻² 10⁻³ (+/−1.7* (n = 1) & 10⁻¹⁰, n = 3) steadystate FGF18 1:1 2.0* 9.1* 4.5* 10⁻⁹ 9.7 6.4*10⁻⁹ 11.8 binding 10⁺⁵ 10⁻³(+/−0.89* & 10⁻⁹, n = 4) steady state Endocrine FGFs FGF19 2:1 5.4* 7.3*1.5* 3.6* 4.8* 10⁻⁷ 0.05 binding 10⁺⁴ 10⁻³ 10⁻¹ 10⁻³ (+/−3.2* 10⁻⁷, n=3) FGF21 2:1 258 1.8* 5.5* 1.4* 2.8* 10⁻⁵ 0.56 binding 10⁻² 10⁻³ 10⁻³(n =2)

For FGF2 and FGF18, a good fit was achieved with a 1:1 binding model,which is the most direct model of binding affinity. This model describesa 1:1 binding interaction at the surface of the chip with immobilizedSFGFR3_DEL4-D3 binding different FGFs: A+B=AB with single on- and offrate. The 2:1 model also describes a 1:1 interaction of FGF binding toSFGFR3_DEL4-D3, but also assumes a conformational change that stabilizesthe complex: A+B=AB=AB* and represents two on- and off-rates. This modelassumes that the conformationally changed complex (SFGFR3_DEL4-D3 boundto FGF) can only dissociate by reversing the conformational change. Theexperimental data for hFGF1, hFGF9, hFGF19, and hFGF21 were determinedto fit the 2:1 model very well, and thus, K_(d) for hFGF1, hFGF9,hFGF19, and hFGF21 were derived from the 2:1 model.

Despite hFGF1, hFGF9, hFGF19, and hFGF21 all having a K_(d) in the lownM range, the kinetic profiles of these hFGFs differed significantly.For example, FGF1 binds sFGFR3_Del4-D3 with a very fast on-rate andoff-rate, while FGF2 does not bind sFGFR3_Del4-D3 with as fast of anon-rate or off-rate as FGF1, resulting in an overall smaller K_(d) forFGF2 compared to FGF1 (Table 8). A significantly lower affinity wasmeasured between sFGFR3_Del4-D3 and hFGF19 or hFGF21, which are membersof the endocrine FGF15/FGF19 subfamily, relative to the paracrine hFGFs(Table 8 and FIGS. 19D and 19E). The FGF15/FGF19 subfamily uses Klothoinstead of proteoglycans as a co-factor and has evolved intoendocrine-acting growth factors, which are important for the systemicregulation of metabolic parameters, such as phosphate, bile acid,carbohydrate, and lipid metabolism.

These results demonstrate that there was a high affinity interaction ofsFGFR3_Del4-D3 with hFGF1, hFGF2, hFGF9, and hFGF18, while there was alow affinity interaction of sFGFR3_Del4-D3 with FGF19 and FGF21. The lowaffinity of sFGFR3_Del4-D3 for FGF19 and FGF21 is advantageous assFGFR3_Del4-D3 will have a low probability of interfering with thefunction of these FGFs in vivo.

Example 24: In Vitro Proliferation Assay of sFGFR3_Del4-D3

Following binding of FGFs, FGFR3 dimerizes to initiate signalingcascades. Several downstream signaling pathways are associated with FGFsignaling. In chondrocytes, dimerized FGFR3 results in ananti-proliferative signal/early differentiation signal into thechondrocyte, which eventually leads to inhibition of bone growth. Forexample, the RAS/MAPK pathway propagates signals to negatively affectproliferation, terminal differentiation, and post-mitotic matrixsynthesis, and the STAT1 pathway mediates the inhibition of chondrocyteproliferation in concert with the cell cycle regulators p107 and 130 andcell cycle inhibitor p21Waf/Cip1. Gene expression studies suggest anumber of other pathways are also involved in down-regulation ofgrowth-promoting molecules or induction of anti-proliferative functions.

To study FGFR3-decoy induced inhibition of FGFR3^(G380R) in achondrocytic cell model, studies were performed to determine the effectof sFGFR3_Del4-D3 on the proliferation of ATDC5 cells geneticallymodified to overexpress the FGFR3^(ach) mutation (ATDC5 FGFR3^(G380R)cells). The chondrocytic cell line ATDC5 cell, which was first isolatedfrom the differentiating teratocarcinoma stem cell line AT805, iscommonly used as a model for in vitro chondrocyte research. ATDC5 cellswere first infected with a retroviral expression vector and a stablecell line expressing FGFR3^(G380R) was generated. The expression ofFGFR3^(G380R) in the ATDC5 cell line was determined via Western blot(FIG. 20). Extracts of ATDC5 cells expressing FGFR3^(G380R) at passageone (G380R #1) and two (G380R #2) after resistant cell selection andextracts of control ATDC5 cells were blotted and detected withantibodies for total phosphorylation of FGFR3 (pFGFR3), the specificphosphotyrosine 724 in FGFR3 (pFGFR3 Y724), and total FGFR3 expression(FGFR3). Total extracellular signal-related kinase expression was usedas loading control (ERK). Addition of SFGFR3_DEL4-D3 to the ATDC5FGFR3^(G380R) cells dose-dependently increased the proliferation indexof the ATDC5 FGFR3^(G380R) cells by two-fold with an EC₅₀ of 1.25+/−0.27nM (FIG. 21). These results demonstrate that addition of SFGFR3_DEL4-D3to ATDC5 FGFR3^(G380R) cells overcomes the negative growth signalmediated by FGFR3^(G380R) in a cellular model of achondroplasia and arein line with the anti-proliferative signal mediated by FGFR3 inchondrocytes, which is more pronounced when the chondrocytes express aFGFR3 including the G380R mutation.

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety. Various modifications and variations of thedescribed methods, pharmaceutical compositions, and kits of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it will beunderstood that it is capable of further modifications and that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the invention. This applicationis intended to cover any variations, uses, or adaptations of theinvention following, in general, the principles of the invention andincluding such departures from the present disclosure come within knowncustomary practice within the art to which the invention pertains andmay be applied to the essential features herein before set forth.

1.-30. (canceled)
 31. A polynucleotide encoding a soluble fibroblastgrowth factor receptor 3 (sFGFR3) polypeptide comprising the amino acidsequence of SEQ H) NO:
 33. 32. The polynucleotide of claim 31, whereinthe polynucleotide comprises a nucleic acid sequence at least 80%identical to nucleotides 58-1104 of SEQ ID NO:
 37. 33. Thepolynucleotide of claim 31, wherein the polynucleotide comprises thenucleic acid sequence of nucleotides 58-1104 of SEQ ID NO:
 37. 34. Thepolynucleotide of claim 31, wherein the polynucleotide comprises anucleic acid sequence at least 80% identical to nucleotides 52-1098 ofSEQ II) NO:
 21. 35. The polynucleotide of claim 31, wherein thepolynucleotide comprises the nucleic acid sequence of nucleotides52-1098 of SEQ ID NO:
 21. 36. The polynucleotide of claim 31, whereinthe sFGFR3 polypeptide hinds to fibroblast growth factor 1 (FGF1),fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (RiF9),fibroblast growth factor 18 (FGF18), fibroblast growth factor 19(FGF19), or fibroblast growth factor 2.1 (FGF21).
 37. The polynucleotideof claim 36, wherein the binding is characterized by a K_(d) of about 1nM to about 10 nM.
 38. A vector comprising the polynucleotide of claim31.
 39. The vector of claim 38, wherein vector comprises the nucleicacid sequence of nucleotides 58-1104 of SEQ ID NO:
 37. 40. The vector ofclaim 38, wherein the vector comprises the nucleic acid sequence of SEQID NO:
 37. 41. The vector of claim 37, wherein vector comprises thenucleic acid sequence of nucleotides 52-1098 of SEQ ID NO:
 21. 42. Thevector of claim 38, wherein the vector is a plasmid, an artificialchromosome, a viral vector or a phage vector.
 43. A method of producinga soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide, themethod comprising: (i) culturing a host cell comprising thepolynucleotide of claim 31 in a culture medium under conditions suitablefor expression of the sFGFR3 polypeptide; and (ii) recovering the sFGFR3polypeptide from the culture medium.
 44. The method of claim 43, whereinthe polynucleotide comprises the nucleic acid sequence of nucleotides58-1104 of SEQ H) NO: 37, or the nucleic acid sequence of nucleotides52-1098 of SEQ ID NO:
 21. 45. The method of claim 43, wherein the hostcell is HEK293 cell or CHO cell.
 46. A method of producing a solublefibroblast growth factor receptor 3 (sFGFR3) polypeptide, the methodcomprising: (i) culturing a host cell comprising the vector of claim 38in a culture medium under conditions suitable for expression of thesFGFR3 polypeptide; and (ii) recovering the sFGFR3 polypeptide from theculture medium.
 47. The method of claim 46, wherein the vector comprisesthe nucleic acid sequence of nucleotides 58-1104 of SEQ NO: 37, or thenucleic acid sequence of nucleotides 52-1098 of SEQ ID NO:
 21. 48. Themethod of claim 46, wherein the vector comprises the nucleic acidsequence of SEQ ID NO:
 37. 49. The method of claim 46, wherein the hostcell is HEK293 cell or CHO cell.